Ligand activated transcriptional regulator proteins

ABSTRACT

Fusion proteins for use as ligand-dependent transcriptional regulators are provided. The fusion proteins include a nucleotide binding domain operatively linked to a ligand-binding domain. They also can include a transcription regulating domain. The nucleotide binding domain is a zinc-finger peptide that binds to a targeted contiguous nucleotide sequence of from 3 to about 18 nucleotides are provided. The fusion proteins are used for gene therapy. Also provided are polynucleotides encoding the fusion proteins, expression vectors, and transfected cells.

RELATED APPLICATIONS

This application is a divisional of allowed U.S. application Ser. No.10/422,934, filed Apr. 23, 2003, which is a continuation of U.S.application Ser. No. 09/586,625, filed Jun. 2, 2000, now U.S. Pat. No.7,329,728, which is a continuation-in-part of U.S. application Ser. No.09/433,042, filed Oct. 25, 1999, now abandoned. The subject matter ofeach of U.S. application Ser. Nos. 09/433,042, 09/586,625 and 10/422,934is herein incorporated by reference in its entirety.

Work described herein was supported by National Institutes of Health NIHContract No. GM53910. The United States Government has certain rights insuch subject matter.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Jul. 18, 2008, is identical, 234 kilobytes in size, andtitled 1227DSEQ.001.txt.

FIELD OF THE INVENTION

The field of this invention is the regulation of gene expression. Inparticular, ligand-activated fusion proteins (also referred to herein aschimeric regulators) and the use thereof for regulation of geneexpression are provided. The fusion polypeptides contain a DNA bindingdomain containing one or a plurality of zinc finger polypeptide domainsand a ligand binding domain (LBD) derived from an intracellularreceptor.

BACKGROUND OF THE INVENTION

Intracellular receptors are a superfamily of related proteins thatmediate the nuclear effects of a variety of hormones and effectormolecules, including steroid hormones, thyroid hormones and vitamins Aand D. Members of this family of intracellular receptors areprototypical ligand activated transcription factors. These receptorscontain two primary functional domains: a DNA binding domain (DBD) thatcontains about sixty-six amino acids and a ligand-binding domain (LBD)located in the carboxyl-terminal half of the receptor that has about 300amino acids. The receptors are inactive in the absence of hormone(ligand) by virtue of association with inactivating factors, such asheat shock proteins. Upon ligand binding, the receptors dissociate fromthe inactivating complex and dimerize, which renders them able to bindto DNA and modulate transcription.

For example, for the steroid receptors, binding of a steroid hormone toits receptor results in receptor protein homodimerization and subsequentbinding to the “steroid response element” (SRE) DNA sequence in nuclearDNA. Conformational changes in the receptor associated with ligandbinding results in the recruitment of other transcriptional regulatoryproteins, called co-activators, that regulate the transcription frompromoters adjacent to the SRE binding sites.

Modified steroid hormone receptors have been developed for use forregulated expression of transgenes (see, e.g., U.S. Pat. No. 5,874,534and published International PCT application No. WO 98/18925, which isbased on U.S. provisional application Ser. No. 60/029,964) by modifyingthe ligand specificity of the LBD. In addition, the DNA binding domainof the receptor has been replaced with a non-mammalian DNA bindingdomain selected from yeast GAL4 DBD, a viral DBD and an insect DBD toprovide for regulated expression of a co-administered gene containing aregion recognized by the non-mammalian DBD. These constructs, however,have several drawbacks. The non-mammalian DBD is potentially immunogenicand the array of sequences recognized by these DBD is limited, therebyseverely restricting gene targets.

Therefore, there remains a need for more versatile gene regulators. Itis an object herein to provide polypeptides that function as versatileregulators of gene expression.

SUMMARY OF THE INVENTION

Polypeptides that function as ligand activated transcriptionalregulators and nucleic acid molecules encoding such polypeptides areprovided. The polypeptides are fusion proteins that are ligand activatedtranscriptional regulators that can be targeted to any desiredendogenous or exogenous gene. Variants of the fusion protein can bedesigned to have different selectivity and sensitivity for endogenousand exogenous ligands.

Nucleic acid molecules encoding the fusion proteins, expression vectorscontaining the nucleic acids and cells containing the expression vectorsare provided. The fusion protein or nucleic acids, particularly vectors,that encode the fusion protein can be introduced into a cell and, whenexpressed in the cell, regulate gene expression in a ligand-dependentmanner.

Fusion Proteins

The fusion proteins provided herein contain a ligand binding domain(designated herein LBD) from an intracellular receptor, preferably a LBDthat has modified ligand specificity compared to the nativeintracellular receptor from which the LBD originates, and a nucleic acidbinding domain (designated herein DBD) that can be tailored for anydesired specificity. The fusion proteins may also include atranscriptional regulating domain (designated herein TRD), particularlya repressor or activator domain. The domains are operatively linkedwhereby the resulting fusion protein functions as a ligand-regulatedtargeted transcription factor.

When delivered to the nucleus of a cell, the domains, which areoperatively linked, together act to modulate the expression of atargeted gene, which may be a native gene in a cell or a gene that alsois delivered to a cell. Hence the targeted gene can be an endogenouscellular gene or an exogenously supplied recombinant polynucleotideconstruct. The fusion protein may also include a transcriptionalregulating domain that is selected to activate, enhance or suppresstranscription of a targeted gene.

In one embodiment, the fusion protein is constructed from componentshighly similar to human proteins, preferably components that are about80% more preferably about 85%, most preferably at least about 90%identical in amino acid sequence to the corresponding human domain. Inanother embodiment, the fusion protein binds to a naturally occurringgene and modulates the transcription of the naturally occurring gene ina ligand-dependent way. In another embodiment, the fusion protein bindsto an exogenously supplied recombinant construct and modulates thetranscription of the exogenously supplied recombinant construct in aligand-dependent way.

In a preferred embodiment, the isolated recombinant fusion protein formsa dimer when bound to a polynucleotide. The dimer can be a homodimer ora heterodimer. In one embodiment, the dimer includes at least one DNAbinding domain, at least one, preferably two, ligand binding domains andat least one transcription modulating domain. In heterodimers, the dimercan include two different DNA binding domains, two different ligandbinding domains or two different transcription modulating domains. Oneexemplary heterodimer includes at least three zinc finger modular units,two different ligand binding sites and a transcription modulatingdomain.

Exemplary fusion proteins containing zinc fingers and LBD that arenon-responsive to estrogen, and that are induced by syntheticnon-steroidal drugs that are routinely used for clinical treatments aredescribed; these regulators provide ligand-dependent gene activation.Exemplary fusion proteins comprise the sequence of amino acids encodedby the open reading frame set forth in each of SEQ ID Nos. 1-18.

The fusion proteins can be used in plant species as well as animals.Transgenic plants resistant to particular bacterial or viral pathogenscan be produced.

Ligand Binding Domain (LBD)

The LBD is derived from an intracellular receptor, particularly asteroid hormone receptor. The receptors from which the LBD is derivedinclude, but is not limited to, glucocorticoid receptors,mineralocorticoid receptors, thyroid hormone receptors, retinoic acidreceptors, retinoid X receptors, Vitamin D receptors, COUP-TF receptors,ecdysone receptors, Nurr-1 receptors, orphan receptors and variantsthereof. Receptors of these types include, but are not limited to,estrogen receptors, progesterone receptors, glucocorticoid-α receptors,glucocorticoid-β receptors, androgen receptors and thyroid hormonereceptors. LBDs preferably are modified to alter ligand specificity sothat they preferentially bind to an exogenous ligand, such as a drug,compared to an endogenous ligand.

When intended for human gene therapy, the ligand binding domainpreferably retain sufficient identity, typically at least about 90%sequence identity to a human ligand binding domain, to avoid substantialimmunological response. A single amino acid change in the LBD candramatically alter performance of the protein.

The LBD is preferably modified so that it does not bind to theendogenous ligand for the receptor from which the LBD is derived, but toa selected ligand to permit fine tuned regulation of targeted genes.Hence, in certain embodiments, the ligand-binding domain has beenmodified to change its ligand selectivity compared to its selectivity inthe native receptor. Preferably the modified ligand-binding domain isnot substantially activated by endogenous ligands. Any method foraltering ligand specificity, including systematic sequence alterationand testing for specificity, and selection protocols (see, e.g., U.S.Pat. No. 5,874,534 and Wang et al. (1994) Proc. Natl. Acad. Sci. U.S.A.91:8180-8184) can be used.

Nucleic Acid Binding Domain (DBD)

To achieve targeted and specific transcriptional regulation the DBDincludes at least one zinc finger modular unit and is engineered to bindto targeted genes. The zinc finger nucleic acid binding domain containsat least two zinc finger modules that bind to selected sequences ofnucleotides. Any zinc finger or modular portions thereof can be used.The DBD replaces or supplements the naturally-occurring zinc fingerdomain in the receptor from which the ligand binding domain is derived.

The nucleic acid binding domain (DBD) includes at least one, preferablyat least two, modular units of a zinc finger nucleic acid bindingpolypeptide, each modular unit specifically recognizing a threenucleotide sequence of bases. The resulting DBD binds to a contiguoussequence of nucleotides of from 3 to about 18 nucleotides.

As noted, the DBD contains modular zinc-finger units, where each unit isspecific for a trinucleotide. Modular zinc protein units can be combinedso that the resulting domain specifically binds to any targetedsequence, generally DNA, such that upon binding of the fusion protein tothe targeted sequence transcription of the targeted gene is modulated.

The zinc finger-nucleotide binding portion of the fusion protein can bederived or produced from a wild type zinc finger protein by truncationor expansion, or as a variant of a wild type-derived polypeptide by aprocess of site directed mutagenesis, or by combination of a variety ofmodular units or by a combination of procedures.

Cys₂His₂ (C2H2) type zinc finger proteins are exemplary of the zincfingers that can replace the naturally occurring DNA binding domain inan intracellular receptor, such as the C4-C4 type domain in a steroidreceptor, to form a functional ligand-responsive transcription factorfusion protein. By virtue of the zinc finger, the resulting fusionprotein exhibits altered DNA binding specificity compared to theunmodified intracellular receptor.

The optimal portion of the ligand binding domain (LBD) of the receptorto use, the zinc finger array and extent thereof and the stoichiometryand orientation of DNA binding can be empirically determined asexemplified herein for a steroid receptor.

In preferred embodiments the zinc-finger portion of the fusion proteinbinds to a nucleotide sequence of the formula (GNN)_(n), where G isguanidine, N is any nucleotide and n is an integer from 1 to 6, andtypically n is 3 to 6. Preferably, the zinc-finger modular unit isderived from C2H2 zinc-finger peptide. More preferably, the zinc-fingerpeptide is a C2H2 zinc-finger peptide and has at least 90% sequenceidentity to a human zinc-finger peptide.

Transcription Regulating Domain (TRD)

The fusion proteins also can include transcription regulating domains.In preferred embodiments, the transcription regulating domain includes atranscription activation domain. Preferably, the transcriptionregulating domain has at least 90% sequence identity to a mammalian,including human if the fusion protein is intended for human genetherapy, transcription regulating domain to avoid inducing undesirableimmunological responses.

The transcription regulating domain can be any such domain known toregulate or prepared to regulate eukaryotic transcription. Such TRDs areknown, and include, but are not limited to, VP16, VP64, TA2, STAT-6,p65, and derivatives, multimers and combinations thereof that exhibittranscriptional regulation properties. The transcription regulatingdomain can be derived from an intracellular receptor, such as a nuclearhormone receptor transcription activation (or repression) domain, and ispreferably a steroid hormone receptor transcription activation domain orvariant thereof that exhibits transcriptional regulation properties.Transcription domains include, but are not limited to, TAF-1, TAF-2,TAU-1, TAU-2, and variants thereof.

The transcription regulating domain may be a viral transcriptionactivation domain or variant thereof. Preferably, the viraltranscription regulating domain comprises a VP16 transcriptionactivation domain or variant thereof.

The transcription regulating domain can include a transcriptionrepression domain. Such domains are known, and include, but are notlimited to, transcription repression domains selected from among ERD,KRAB, SID, Deacetylase, and derivatives, multimers and combinationsthereof, such as KRAB-ERD, SID-ERD, (KRAB)₂, (KRAB)₃, KRAB-A, (KRAB-A)₂,(SID)₂ (KRAB-A)-SID and SID-(KRAB-A).

Nucleic Acid Constructs

Also provided are nucleic acid molecules that encode the resultingfusion proteins. The nucleic acids can be included in vectors, suitablefor expression of the proteins and/or vectors suitable for gene therapy.Cells containing the vectors are also provided. Typically the cell is aeukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

Also provided are expression cassettes that contain a gene of interest,particularly a gene encoding a therapeutic product, such as anangiogenesis inhibitor, operatively linked to a transcriptionalregulatory region or response element, including sequences of nucleicacids to which a fusion protein provided herein binds and controlstranscription, particularly upon binding of a ligand to the LBD of thefusion polypeptide. Such expression cassettes can be included in avector for gene therapy, and are intended for administration with,before or after, administration of the fusion protein or nucleic acidencoding the fusion protein. Genes of interest for exogenous deliverytypically encode therapeutic proteins, such as growth factors, growthfactor inhibitors or antagonists, tumor necrosis factor (TNF)inhibitors, anti-tumor agents, angiogenesis agents, anti-angiogenesisagents, clotting factors, apoptotic and other suicide genes.

Compositions, Combinations and Kits

Also provided are compositions that contain the fusion proteins or thevectors that encode the fusion proteins. Combinations of the fusionproteins or nucleic acids encoding the proteins and nucleic acidencoding a targeted gene with regulatory regions selected for activationby the fusion protein are also provided.

Compositions, particularly pharmaceutical compositions containing thefusion polypeptides in a pharmaceutically acceptable carrier are alsoprovided.

Combinations of the expression cassette and fusion polypeptide ornucleic acid molecules, particularly expression vectors that encode thefusion polypeptide are provided. The combinations may include separatecompositions or a single composition containing both elements. Kitscontaining the combinations and optionally instructions foradministration thereof and other reagents used in preparing andadministering the combinations are also provided.

Hence compositions suitable for gene therapy that contain nucleic acidencoding the fusion protein, typically in a vector suitable for genetherapy are provided. Preferred vectors include viral vectors,preferably adenoviral vectors, and lentiviral vectors. In otherembodiments, non-viral delivery systems, including DNA-ligand complexes,adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, liposomes andlipofection are provided.

The compositions suitable for regulating gene expression contain aneffective amount of the fusion protein or a polynucleotide encoding theligand activated transcriptional regulatory fusion protein and apharmaceutically acceptable excipient. Such compositions can furtherinclude a regulatable expression cassette encoding a gene and at leastone response element for the gene recognized by the nucleotide bindingdomain of the fusion polypeptide.

The regulatable expression cassette is designed to include a sequence ofnucleic acids with which the nucleic acid binding domain of the ligandactivated transcriptional regulatory fusion protein interacts. It alsopreferably includes operatively linked transcriptional regulatorysequences that are regulatable by the TRD of the fusion protein.Typically, the regulatable expression cassette includes 3 to 6 responseelements.

Methods

Methods for regulating expression of endogenous and exogenous genes areprovided. The methods are practiced by administering to a cell acomposition that contains an effective amount or concentration of thefusion protein or of nucleic acid molecule, such as a vector thatencodes the fusion protein. The nucleic acid binding domain (DBD) of thefusion protein is selected to bind to a targeted nucleic acid sequencein the genome of the cell or in an exogenously administered nucleic acidmolecule, and the transcription regulating domain (TRD) is selected toregulate transcription from a selected promoter, which typically isoperatively linked to the targeted nucleic acid binding domain. Theexogenously administered nucleic acid molecule comprises an expressioncassette encoding a gene of interest and operatively linked to aregulatory region that contains elements, such as a promoter andresponse elements.

As noted the targeted regulatory region and gene of interest may beendogenously present in the cell or separately administered as part ofan expression cassette encoding the gene of interest. If separatelyadministered, it is administered as part of a regulatable expressioncassette that includes a gene and at least one response element for thegene recognized by the nucleotide binding domain of the fusion protein.

At the same time or at a later time, a composition comprising a ligandthat binds to the ligand binding domain of the fusion protein is alsoadministered. The ligand can be administered in the same composition asthe fusion protein (or encoding nucleic acid molecule) or in a separatecomposition. The ligand and fusion protein may be administeredsequentially, simultaneously or intermittently.

Hence gene therapy is effected by administering a ligand that binds tothe LBD of the fusion protein. Preferably the ligand is a non-naturalligand and the LBD has been modified from the native form present innative intracellular receptors to preferentially and selectivelyinteract with the non-natural ligand. Upon administration, the ligandbinds to the ligand binding domain of the fusion protein, whereby theDBD of the fusion protein, either as a monomer or dimer, interacts witha targeted gene and transcription of the targeted gene is repressed oractivated. As noted, the targeted gene may be an endogenous gene or anexogenously administered gene.

In other embodiments, the methods for regulating gene expression in acell are effected by administering to the cell a composition containingan effective amount of the nucleic acid molecule that encodes the ligandactivated transcriptional regulatory fusion protein, a regulatableexpression cassette containing a gene operatively linked to at least oneresponse element for the gene recognized by the nucleotide bindingdomain of the polypeptide encoded by the polynucleotide, and apharmaceutically acceptable excipient; and administering to the cell aligand that binds to the ligand binding domain of the encodedpolypeptide, where the nucleotide binding domain of the encodedpolypeptide binds to the response element and activates or repressestranscription of the gene.

Methods for treating a cellular proliferative disorder by the ex vivointroduction of a recombinant expression vector encoding the fusionprotein are provided. Cellular proliferative disorders include disordersassociated with transcription of a gene at reduced or increased levels.

Administration of the composition(s) can be effected in vitro, in vivoor ex vivo. One such method includes the removal of a tissue sample froma subject with a disorder, such as a cell proliferative disorder,isolating hematopoietic or other cells from the tissue sample, andcontacting isolated cells with the fusion protein or a nucleic acidmolecule encoding the fusion protein, and, optionally, a target specificgene. Optionally, the cells can be treated with a growth factor, such asinterleukin-2 for example, to stimulate cell growth, beforereintroducing the cells into the subject. When reintroduced, the cellsspecifically target the cell population from which they were originallyisolated. In this way, the trans-repressing activity of the zincfinger-nucleotide binding polypeptide may be used to inhibit or suppressundesirable cell proliferation in a subject. Preferably, the subject isa human.

Results exemplified herein demonstrate ligand activated transcription ofa targeted gene and demonstrate the utility of the fusion proteincontaining a zinc finger DNA binding domain, such as a mammalian C2H2DNA binding domain, a ligand binding domain from an intracellularreceptor, such as an estrogen receptor, and, optionally, a heterologoustranscription regulating domain for the purpose of obtainingligand-dependent control of expression of a transgene introduced intomammalian cells. Hence it is shown herein that heterologous zinc fingerdomains can be combined with an intracellular receptor to achieveligand-dependent gene expression of a targeted gene.

DESCRIPTION OF THE DRAWINGS

In the drawings, which form a portion of the specification:

FIG. 1 is a schematic for the selection strategy for the in vitroevolution of the 3 finger protein Zif268, recognizing its natural 9 bptarget site (top), into a 6 finger protein, recognizing a desired 18 bptarget sequence (bottom).

FIG. 2 is a schematic depiction of the functional domains (A-F) of thehuman estrogen receptor.

FIG. 3 is a schematic depiction of the cloning strategy for theconstruction of the recombinant molecular constructs.

FIG. 4 is a schematic map of the expression vector for C7LBDAS based onthe plasmid pcDNA3.1.

FIG. 5 is a schematic map of the expression vector for C7LBDBS based onthe plasmid pcDNA3.1.

FIG. 6 is a schematic map of the expression vector for C7LBDCS based onthe plasmid pcDNA3.1.

FIG. 7 is a schematic map of the expression vector for C7LBDAL based onthe plasmid pcDNA3.1.

FIG. 8 is a schematic map of the expression vector for C7LBDBL based onthe plasmid pcDNA3.1.

FIG. 9 is a schematic map of the expression vector for C7LBDCL based onthe plasmid pcDNA3.1.

FIG. 10 is a schematic summary of the structure of several embodimentsof the recombinant molecular construct and the nucleotide sequences ofthe DNA binding regions of zinc finger domains C7, E2C and 2C7.

FIG. 11 is a schematic map of the expression vector for E2CLBDAS basedon the plasmid pcDNA3.1.

FIG. 12 is a schematic map of the expression vector for E2CLBDBS basedon the plasmid pcDNA3.1.

FIG. 13 is a schematic diagram of the constructs C7LBDASTA2, C7LBDBSTA2,C7LBDBS-STAT6, C7LBDBSVP16 (SEQ ID NO: 16), AND C7LBDBSNLSVP16.

FIG. 14 is a schematic restriction map of constructs comprising RXR andecdysone (EcR) ligand binding domains used in heterodimers.

FIG. 15 is a schematic depiction of the cloning strategy for theconstruction of the 2C7LBD recombinant molecular constructs.

FIG. 16 is a schematic map of the expression vector for 2C7LBDAS basedon the plasmid pcDNA3.1.

FIG. 17 is a schematic map of the expression vector for 2C7LBDBS basedon the plasmid pcDNA3.1.

FIG. 18 is a schematic map of the expression vector for 2C7LBDCS basedon the plasmid pcDNA3.1.

FIG. 19 is a schematic map of the expression vector for LBDASNLSVP16(SEQ ID NO: 13), based on the plasmid pcDNA3.1.

FIG. 20 is a schematic map of the expression vector for C7LBDBSVP16based on the plasmid pcDNA3.1.

FIG. 21 is a schematic map of the expression vector for C7LBDBSG521R(SEQ ID NO: 15), based on the plasmid pcDNA3.1.

FIG. 22 is a schematic map of the expression vector for C7LBDBSG400V(SEQ ID NO: 14), based on the plasmid pcDNA3.1.

FIG. 23 shows A: an inducible promoter based on binding sites for the 3Finger protein N1. The promoter contains 5 direct repeats of N1 sitesspaced by 3 bp; the spacing between the 5 repeats is 6 bp. Bottom:Luciferase assay. HeLa cells were cotransfected with plasmids encodingthe indicated fusion proteins and the N1 reporter construct. Twenty fourhours later, the cells were treated with 10 nM RU486 (B) or 100 nMTamoxifen, C respectively. Forty-eight hours post transfection, cellextracts were assayed for luciferase activity.

FIG. 24 shows an inducible promoter based on binding sites for the 3Finger protein B3. A: The promoter contains 5 direct repeats of B3 sitesspaced by 3 bp; the spacing between the 5 repeats is 6 bp. Bottom:Luciferase assay. HeLa cells were cotransfected with plasmids encodingthe indicated fusion proteins and the B3 reporter construct. At 24 hlater, the cells were treated with 10 nM RU486 (B), or 100 nM Tamoxifen(C), respectively. At 48 h post transfection, cell extracts were assayedfor luciferase activity.

FIG. 25 is a graphical depiction of the results of luciferase assayshowing the RU486-induced formation of functional VP64-C7-PR/VP64-CF2-PRheterodimers. HeLa cells were cotransfected with the correspondingeffector plasmids and TATA reporter plasmids (C7/CF2-dr0, C7 site 5′ toa CF2 site, direct “repeat”, no spacing; C7/C7-dr0, 2 C7 sites, directrepeat, no spacing). At 24 h later, the cells were treated with 10 nMRU486. At 48 h post transfection, cell extracts were assayed forluciferase activity.

FIG. 26 shows a restriction map for the plasmid designated pAvCVlx.

FIG. 27 shows a restriction map for the plasmid designated pSQ3.

DETAILED DESCRIPTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, applications,published applications and other publications and sequences from GenBankand other data bases referred to anywhere in the disclosure herein areincorporated by reference in their entirety.

As used herein, the ligand binding domain (LBD) of the fusion proteinsprovided herein refers to the portion of the fusion protein responsiblefor binding to a selected ligand. The LBD optionally and preferablyincludes dimerization and inactivation functions. The LBDs in theproteins herein are derived from the 300 amino acid carboxyl-terminalhalf of intracellular receptors, particularly those that are members ofthe steroid hormone nuclear receptor superfamily. It is the portion ofthe receptor protein with which a ligand interacts thereby inducing acascade of events leading to the specific association of an activatedreceptor with regulatory elements of target genes. In these receptorsthe LBD includes the hormone binding function, the inactivationfunction, such as through interactions with heat shock proteins (hsp),and dimerization function. The LBDs used herein include such LBDs andmodified derivatives thereof, particularly forms with altered ligandspecificity.

As used herein, the transcription regulating domain (TRD) refers to theportion of the fusion polypeptide provided herein that functions toregulate gene transcription. Exemplary and preferred transcriptionrepressor domains are ERD, KRAB, SID, Deacetylase, and derivatives,multimers and combinations thereof such as KRAB-ERD, SID-ERD, (KRAB)₂,(KRAB)₃, KRAB-A, (KRAB-A)₂, (SID)₂ (KRAB-A)-SID and SID-(KRAB-A).

As used herein, the DNA binding domain (DBD), or alternatively thenucleic acid (or nucleotide) binding domain, refers to the portion ofthe fusion polypeptide provided herein that provides specific nucleicacid binding capability. The use of the abbreviation DBD is not meant tolimit it to DNA binding domains, but is also intended to includepolypeptides that bind to RNA. The nucleic acid binding domain functionsto target the protein to specific genes by virtue of the specificity ofthe interaction of the TRD region for nucleotide sequences operativelylinked to the transcriptional apparatus of a gene. The DBD targets thefusion protein to the selected targeted gene or genes, which gene(s) maybe endogenous or exogenously added.

As used herein, operatively linked means that elements of the fusionpolypeptide, for example, are linked such that each perform or functionas intended. For example, the repressor is attached to the bindingdomain in such a manner that, when bound to a target nucleotide via thatbinding domain, the repressor acts to inhibit or prevent transcription.Linkage between and among elements may be direct or indirect, such asvia a linker. The elements are not necessarily adjacent. Hence arepressor domain of a TRD can be linked to a DNA binding domain usingany linking procedure well known in the art. It may be necessary toinclude a linker moiety between the two domains. Such a linker moiety istypically a short sequence of amino acid residues that provides spacingbetween the domains. So long as the linker does not interfere with anyof the functions of the binding or repressor domains, any sequence canbe used.

As used herein, a fusion protein is a protein that contains portions orfragments of two or more naturally-occurring proteins operatively joinedor linked to form the fusion protein in which each fragment retains afunction or a modified function exhibited by the naturally occurringproteins. The fragments from the naturally occurring protein may bemodified to alter the original properties.

As used herein, modified, modification, mutant or other such termsrefers to an alteration of the domain in question from its naturallyoccurring wild-type form, and includes primary sequence changes.

As used herein, “modulating” envisions the inhibition or suppression ofexpression from a promoter containing a zinc finger-nucleotide bindingmotif when it is over-activated, or augmentation or enhancement ofexpression from such a promoter when it is underactivated.

As used herein, steroid hormone receptor superfamily refers to thesuperfamily of intracellular receptors that are steroid receptors.Representative examples of such receptors include, but are not limitedto, the estrogen, progesterone, glucocorticoid-α, glucocorticoid-β,mineralocorticoid, androgen, thyroid hormone, retinoic acid, retinoid X,Vitamin D, COUP-TF, ecdysone, Nurr-1 and orphan receptors.

As used herein, the amino acids, which occur in the various amino acidsequences appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations. The nucleotides,which occur in the various DNA fragments, are designated with thestandard single-letter designations used routinely in the art.

In a peptide or protein, suitable conservative substitutions of aminoacids are known to those of skill in this art and may be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224).

As used herein, a delivery plasmid is a plasmid vector that carries ordelivers nucleic acids encoding a therapeutic gene or gene that encodesa therapeutic product or a precursor thereof or a regulatory gene orother factor that results in a therapeutic effect when delivered in vivoin or into a cell line, such as, but not limited to a packaging cellline, to propagate therapeutic viral vectors.

As used herein, “recombinant expression vector” or “expression vector”refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of heterologous DNA, suchas nucleic acid encoding the fusion proteins herein or expressioncassettes provided herein. Such expression vectors contain a promotersequence for efficient transcription of the inserted nucleic acid in acell. The expression vector typically contains an origin of replication,a promoter, as well as specific genes that permit phenotypic selectionof transformed cells.

As used herein, a DNA or nucleic acid homolog refers to a nucleic acidthat includes a preselected conserved nucleotide sequence, such as asequence encoding a therapeutic polypeptide. By the term “substantiallyhomologous” is meant having at least 80%, preferably at least 90%, mostpreferably at least 95% homology therewith or a less percentage ofhomology or identity and conserved biological activity or function.

As used herein, “host cells” are cells in which a vector can bepropagated and its DNA expressed. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Such progeny are included when the term “host cell”is used. Methods of stable transfer where the foreign DNA iscontinuously maintained in the host are known in the art.

The terms “homology” and “identity” are often used interchangeably. Inthis regard, percent homology or identity may be determined, forexample, by comparing sequence information using a GAP computer program.The GAP program uses the alignment method of Needleman and Wunsch((1970) J. Mol. Biol. 48:443), as revised by Smith and Waterman ((1981)Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity asthe number of aligned symbols (i.e., nucleotides or amino acids) whichare similar, divided by the total number of symbols in the shorter ofthe two sequences. The preferred default parameters for the GAP programmay include: (1) a unary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) and the weighted comparison matrixof Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described bySchwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE,National Biomedical Research Foundation, pp. 353-358 (1979); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can bedetermined using known computer algorithms such as the “FAST A” program,using for example, the default parameters as in Pearson et al. (1988)Proc. Natl. Acad. Sci. USA 85:2444. Alternatively the BLAST function ofthe National Center for Biotechnology Information database may be usedto determine identify.

In general, sequences are aligned so that the highest order match isobtained. “Identity” per se has an art-recognized meaning and can becalculated using published techniques (see, e.g.: ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity between two polynucleotide or polypeptidesequences, the term “identity” is well known to skilled artisans(Carillo et al. (1988) SIAM J Applied Math 48:1073). Methods commonlyemployed to determine identity or similarity between two sequencesinclude, but are not limited to, those disclosed in Guide to HugeComputers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, andCarillo et al. (1988) SIAM J Applied Math 48:1073. Methods to determineidentity and similarity are codified in computer programs. Preferredcomputer program methods to determine identity and similarity betweentwo sequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP,BLASTN, and FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. Forexample, a test polypeptide may be defined as any polypeptide that is90% or more identical to a reference polypeptide. As used herein, theterm at least “90% identical to” refers to percent identities from 90 to99.99 relative to the reference polypeptides. Identity at a level of 90%or more is indicative of the fact that, assuming for exemplificationpurposes a test and reference polynucleotide length of 100 amino acidsare compared. No more than 10% (i.e., 10 out of 100) amino acids in thetest polypeptide differs from that of the reference polypeptides.Similar comparisons may be made between a test and referencepolynucleotides. Such differences may be represented as point mutationsrandomly distributed over the entire length of an amino acid sequence orthey may be clustered in one or more locations of varying length up tothe maximum allowable, e.g. 10/100 amino acid difference (approximately90% identity). Differences are defined as nucleic acid or amino acidsubstitutions, or deletions.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, preferably more thanthree, from which synthesis of a primer extension product can beinitiated. For purposes herein, a primer of interest is one that issubstantially complementary to a zinc finger-nucleotide binding proteinstrand, but also can introduce mutations into the amplification productsat selected residue sites. Experimental conditions conducive tosynthesis include the presence of nucleoside triphosphates and an agentfor polymerization and extension, such as DNA polymerase, and a suitablebuffer, temperature and pH.

As used herein, genetic therapy involves the transfer of heterologousDNA to the certain cells, target cells, of a mammal, particularly ahuman, with a disorder or conditions for which such therapy is sought.The DNA is introduced into the selected target cells in a manner suchthat the heterologous DNA is expressed and a therapeutic product encodedthereby is produced. Alternatively, the heterologous DNA may in somemanner mediate expression of DNA that encodes the therapeutic product,or it may encode a product, such as a peptide or RNA that in some mannermediates, directly or indirectly, expression of a therapeutic product.Genetic therapy may also be used to deliver nucleic acid encoding a geneproduct that replaces a defective gene or supplements a gene productproduced by the mammal or the cell in which it is introduced. Theintroduced nucleic acid may encode a therapeutic compound, such as agrowth factor inhibitor thereof, or a tumor necrosis factor or inhibitorthereof, such as a receptor therefor, that is not normally produced inthe mammalian host or that is not produced in therapeutically effectiveamounts or at a therapeutically useful time. The heterologous DNAencoding the therapeutic product may be modified prior to introductioninto the cells of the afflicted host in order to enhance or otherwisealter the product or expression thereof. Genetic therapy may alsoinvolve delivery of an inhibitor or repressor or other modulator of geneexpression.

As used herein, heterologous DNA is DNA that encodes RNA and proteinsthat are not normally produced in vivo by the cell in which it isexpressed or that mediates or encodes mediators that alter expression ofendogenous DNA by affecting transcription, translation, or otherregulatable biochemical processes. Heterologous DNA may also be referredto as foreign DNA. Any DNA that one of skill in the art would recognizeor consider as heterologous or foreign to the cell in which it isexpressed is herein encompassed by heterologous DNA. Examples ofheterologous DNA include, but are not limited to, DNA that encodestraceable marker proteins, such as a protein that confers drugresistance, DNA that encodes therapeutically effective substances, suchas anti-cancer agents, enzymes and hormones, and DNA that encodes othertypes of proteins, such as antibodies. Antibodies that are encoded byheterologous DNA may be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced.

Hence, herein heterologous DNA or foreign DNA, includes a DNA moleculenot present in the exact orientation and position as the counterpart DNAmolecule found in the genome. It may also refer to a DNA molecule fromanother organism or species (i.e., exogenous).

As used herein, a therapeutically effective product is a product that isencoded by heterologous nucleic acid, typically DNA, that, uponintroduction of the nucleic acid into a host, a product is expressedthat ameliorates or eliminates the symptoms, manifestations of aninherited or acquired disease or that cures the disease.

Typically, DNA encoding a desired gene product is cloned into a plasmidvector and introduced by routine methods, such as calcium-phosphatemediated DNA uptake (see, (1981) Somat. Cell. Mol. Genet. 7:603-616) ormicroinjection, into producer cells, such as packaging cells. Afteramplification in producer cells, the vectors that contain theheterologous DNA are introduced into selected target cells.

As used herein, an expression or delivery vector refers to any plasmidor virus into which a foreign or heterologous DNA may be inserted forexpression in a suitable host cell—i.e., the protein or polypeptideencoded by the DNA is synthesized in the host cell's system. Vectorscapable of directing the expression of DNA segments (genes) encoding oneor more proteins are referred to herein as “expression vectors.” Alsoincluded are vectors that allow cloning of cDNA (complementary DNA) frommRNAs produced using reverse transcriptase.

As used herein, a gene refers to a nucleic acid molecule whosenucleotide sequence encodes an RNA or polypeptide. A gene can be eitherRNA or DNA. Genes may include regions preceding and following the codingregion (leader and trailer) as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, isolated with reference to a nucleic acid molecule orpolypeptide or other biomolecule means that the nucleic acid orpolypeptide has separated from the genetic environment from which thepolypeptide or nucleic acid were obtained. It may also mean altered fromthe natural state. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Thus, apolypeptide or polynucleotide produced and/or contained within arecombinant host cell is considered isolated. Also intended as an“isolated polypeptide” or an “isolated polynucleotide” are polypeptidesor polynucleotides that have been purified, partially or substantially,from a recombinant host cell or from a native source. For example, arecombinantly produced version of a compound can be substantiallypurified by the one-step method described in Smith et al. (1988) Gene67:31-40. The terms isolated and purified are sometimes usedinterchangeably.

Thus, by “isolated” the nucleic acid is free of the coding sequences ofthose genes that, in a naturally-occurring genome immediately flank thegene encoding the nucleic acid of interest. Isolated DNA may besingle-stranded or double-stranded, and may be genomic DNA, cDNA,recombinant hybrid DNA, or synthetic DNA. It may be identical to anative DNA sequence, or may differ from such sequence by the deletion,addition, or substitution of one or more nucleotides.

Isolated or purified as it refers to preparations made from biologicalcells or hosts means any cell extract containing the indicated DNA orprotein including a crude extract of the DNA or protein of interest. Forexample, in the case of a protein, a purified preparation can beobtained following an individual technique or a series of preparative orbiochemical techniques and the DNA or protein of interest can be presentat various degrees of purity in these preparations. The procedures mayinclude for example, but are not limited to, ammonium sulfatefractionation, gel filtration, ion exchange chromatography, affinitychromatography, density gradient centrifugation and electrophoresis.

A preparation of DNA or protein that is “substantially pure” or“isolated” should be understood to mean a preparation free fromnaturally occurring materials with which such DNA or protein is normallyassociated in nature. “Essentially pure” should be understood to mean a“highly” purified preparation that contains at least 95% of the DNA orprotein of interest.

A cell extract that contains the DNA or protein of interest should beunderstood to mean a homogenate preparation or cell-free preparationobtained from cells that express the protein or contain the DNA ofinterest. The term “cell extract” is intended to include culture media,especially spent culture media from which the cells have been removed.

As used herein, “modulate” refers to the suppression, enhancement orinduction of a function. For example, zinc finger-nucleic acid bindingdomains and variants thereof may modulate a promoter sequence by bindingto a motif within the promoter, thereby enhancing or suppressingtranscription of a gene operatively linked to the promoter cellularnucleotide sequence. Alternatively, modulation may include inhibition oftranscription of a gene where the zinc finger-nucleotide bindingpolypeptide variant binds to the structural gene and blocks DNAdependent RNA polymerase from reading through the gene, thus inhibitingtranscription of the gene. The structural gene may be a normal cellulargene or an oncogene, for example. Alternatively, modulation may includeinhibition of translation of a transcript.

As used herein, “inhibit” refers to the suppression of the level ofactivation of transcription of a structural gene operably linked to apromoter. For example, for the methods herein the gene includes a zincfinger-nucleotide binding motif.

As used herein, a transcriptional regulatory region refers to a regionthat drives gene expression in the target cell. Transcriptionalregulatory regions suitable for use herein include but are not limitedto the human cytomegalovirus (CMV) immediate-early enhancer/promoter,the SV40 early enhancer/promoter, the JC polyomavirus promoter, thealbumin promoter, PGK and the α-actin promoter coupled to the CMVenhancer.

As used herein, a promoter region of a gene includes the regulatoryelements that typically lie 5′ to a structural gene. If a gene is to beactivated, proteins known as transcription factors attach to thepromoter region of the gene. This assembly resembles an “on switch” byenabling an enzyme to transcribe a second genetic segment from DNA intoRNA. In most cases the resulting RNA molecule serves as a template forsynthesis of a specific protein; sometimes RNA itself is the finalproduct. The promoter region may be a normal cellular promoter or, forexample, an onco-promoter. An onco-promoter is generally a virus-derivedpromoter. Viral promoters to which zinc finger binding polypeptides maybe targeted include, but are not limited to, retroviral long terminalrepeats (LTRs), and Lentivirus promoters, such as promoters from humanT-cell lymphotrophic virus (HTLV) 1 and 2 and human immunodeficiencyvirus (HIV) 1 or 2.

As used herein, “effective amount” includes that amount that results inthe deactivation of a previously activated promoter or that amount thatresults in the inactivation of a promoter containing a zincfinger-nucleotide binding motif, or that amount that blockstranscription of a structural gene or translation of RNA. The amount ofzinc finger derived-nucleotide binding polypeptide required is thatamount necessary to either displace a native zinc finger-nucleotidebinding protein in an existing protein/promoter complex, or that amountnecessary to compete with the native zinc finger-nucleotide bindingprotein to form a complex with the promoter itself. Similarly, theamount required to block a structural gene or RNA is that amount whichbinds to and blocks RNA polymerase from reading through on the gene orthat amount which inhibits translation, respectively. Preferably, themethod is performed intracellularly. By functionally inactivating apromoter or structural gene, transcription or translation is suppressed.Delivery of an effective amount of the inhibitory protein for binding toor “contacting” the cellular nucleotide sequence containing the zincfinger-nucleotide binding protein motif, can be accomplished by one ofthe mechanisms described herein, such as by retroviral vectors orliposomes, or other methods well known in the art.

As used herein, “truncated” refers to a zinc finger-nucleotide bindingpolypeptide derivative that contains less than the full number of zincfingers found in the native zinc finger binding protein or that has beendeleted of non-desired sequences. For example, truncation of the zincfinger-nucleotide binding protein TFIIIA, which naturally contains ninezinc fingers, might be a polypeptide with only zinc fingers one throughthree. Expansion refers to a zinc finger polypeptide to which additionalzinc finger modules have been added. For example, TFIIIA may be extendedto 12 fingers by adding 3 zinc finger domains. In addition, a truncatedzinc finger-nucleotide binding polypeptide may include zinc fingermodules from more than one wild type polypeptide, thus resulting in a“hybrid” zinc finger-nucleotide binding polypeptide.

As used herein, “mutagenized” refers to a zinc finger derived-nucleotidebinding polypeptide that has been obtained by performing any of theknown methods for accomplishing random or site-directed mutagenesis ofthe DNA encoding the protein. For instance, in TFIIIA, mutagenesis canbe performed to replace nonconserved residues in one or more of therepeats of the consensus sequence. Truncated zinc finger-nucleotidebinding proteins can also be mutagenized.

As used herein, a polypeptide “variant” or “derivative” refers to apolypeptide that is a mutagenized form of a polypeptide or one producedthrough recombination but that still retains a desired activity, such asthe ability to bind to a ligand or a nucleic acid molecule or tomodulate transcription.

As used herein, a zinc finger-nucleotide binding polypeptide “variant”or “derivative” refers to a polypeptide that is a mutagenized form of azinc finger protein or one produced through recombination. A variant maybe a hybrid that contains zinc finger domain(s) from one protein linkedto zinc finger domain(s) of a second protein, for example. The domainsmay be wild type or mutagenized. A “variant” or “derivative” includes atruncated form of a wild type zinc finger protein, which contains lessthan the original number of fingers in the wild type protein. Examplesof zinc finger-nucleotide binding polypeptides from which a derivativeor variant may be produced include TFIIIA and zif268. Similar terms areused to refer to “variant” or “derivative” nuclear hormone receptors and“variant” or “derivative” transcription effector domains.

As used herein a “zinc finger-nucleotide binding motif” refers to anytwo or three-dimensional feature of a nucleotide segment to which a zincfinger-nucleotide binding derivative polypeptide binds with specificity.Included within this definition are nucleotide sequences, generally offive nucleotides or less, as well as the three dimensional aspects ofthe DNA double helix, such as, but are not limited to, the major andminor grooves and the face of the helix. The motif is typically anysequence of suitable length to which the zinc finger polypeptide canbind. For example, a three finger polypeptide binds to a motif typicallyhaving about 9 to about 14 base pairs. Preferably, the recognitionsequence is at least about 16 base pairs to ensure specificity withinthe genome. Therefore, zinc finger-nucleotide binding polypeptides ofany specificity are provided. The zinc finger binding motif can be anysequence designed empirically or to which the zinc finger protein binds.The motif may be found in any DNA or RNA sequence, including regulatorysequences, exons, introns, or any non-coding sequence.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike which would be to a degree that would prohibit administration ofthe composition.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. Preferred vectorsare those capable of autonomous replication and expression of structuralgene products present in the DNA segments to which they are operativelylinked. Vectors, therefore, preferably contain the replicons andselectable markers described earlier.

As used herein with regard to nucleic acid molecules, including DNAfragments, the phrase “operatively linked” means the sequences orsegments have been covalently joined, preferably by conventionalphosphodiester bonds, into one strand of DNA, whether in single ordouble stranded form such that operatively linked portions function asintended. The choice of vector to which a transcription unit or acassette provided herein is operatively linked depends directly, as iswell known in the art, on the functional properties desired, e.g.,vector replication and protein expression, and the host cell to betransformed, these being limitations inherent in the art of constructingrecombinant DNA molecules.

As used herein, a sequence of nucleotides adapted for directionalligation, i.e., a polylinker, is a region of the DNA expression vectorthat (1) operatively links for replication and transport the upstreamand downstream translatable DNA sequences and (2) provides a site ormeans for directional ligation of a DNA sequence into the vector.Typically, a directional polylinker is a sequence of nucleotides thatdefines two or more restriction endonuclease recognition sequences, orrestriction sites. Upon restriction cleavage, the two sites yieldcohesive termini to which a translatable DNA sequence can be ligated tothe DNA expression vector. Preferably, the two restriction sitesprovide, upon restriction cleavage, cohesive termini that arenon-complementary and thereby permit directional insertion of atranslatable DNA sequence into the cassette. In one embodiment, thedirectional ligation means is provided by nucleotides present in theupstream translatable DNA sequence, downstream translatable DNAsequence, or both. In another embodiment, the sequence of nucleotidesadapted for directional ligation comprises a sequence of nucleotidesthat defines multiple directional cloning means. Where the sequence ofnucleotides adapted for directional ligation defines numerousrestriction sites, it is referred to as a multiple cloning site.

As used herein, a secretion signal is a leader peptide domain of aprotein that targets the protein to the periplasmic membrane of gramnegative bacteria. A preferred secretion signal is a pelB secretionsignal. The predicted amino acid residue sequences of the secretionsignal domain from two pelB gene product variants from Erwinia carotovaare described in Lei, et al. (Nature, 331:543-546, 1988). The leadersequence of the pelB protein has previously been used as a secretionsignal for fusion proteins (Better et al. (1988) Science 240:1041-1043;Sastry et al. (1989) Proc. Natl. Acad. Sci. USA 86:5728-5732; andMullinax et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8095-8099). Aminoacid residue sequences for other secretion signal polypeptide domainsfrom E. coli are known (see, e.g., Oliver, In Neidhard, F. C. (ed.),Escherichia coli and Salmonella Typhimurium, American Society forMicrobiology, Washington, D.C., 1:56-69 (1987)).

As used herein, ligand refers to any compound that interacts with theligand binding domain of a receptor and modulates its activity; ligandstypically activate receptors. Ligand can also include compounds thatactivate the receptor without binding. A natural ligand is a compoundthat normally interacts with the receptor.

As used herein, anti-hormones are compounds that are antagonists of thenaturally-occurring receptor. The anti-hormone is opposite in activityto a hormone.

As used herein, non-natural ligands or non-native ligands refer tocompounds that are normally not found in mammals, such as humans, thatbind to or interact with the ligand binding domain of a receptor. Hence,the term “non-native ligands” refers to those ligands that are notnaturally found in the specific organism (man or animal) in which genetherapy is contemplated. For example, certain insect hormones such asecdysone are not found in humans. As such ecdysone is non-native hormoneto an animal, such as a human.

As used herein, “cell-proliferative disorder” denotes malignant as wellas non-malignant disorders in which cell populations morphologicallyappear to differ from the surrounding tissue. The cell-proliferativedisorder may be a transcriptional disorder that results in an increaseor a decrease in gene expression level. The cause of the disorder may beof cellular origin or viral origin. Gene therapy using a zincfinger-nucleotide binding polypeptide can be used to treat avirus-induced cell proliferative disorder in a human, for example, aswell as in a plant. Treatment can be prophylactic in order to make aplant cell, for example, resistant to a virus, or therapeutic, in orderto ameliorate an established infection in a cell, by preventingproduction of viral products.

As used herein, “cellular nucleotide sequence” refers to a nucleotidesequence that is present within a cell. It is not necessary that thesequence be a naturally occurring sequence of the cell. For example, aretroviral genome that is integrated within a host's cellular DNA, wouldbe considered a “cellular nucleotide sequence”. The cellular nucleotidesequence can be DNA or RNA and includes introns and exons, DNA and RNA.The cell and/or cellular nucleotide sequence can be prokaryotic oreukaryotic, including a yeast, virus, or plant nucleotide sequence.

As used herein, administration of a therapeutic composition can beeffected by any means, and includes, but is not limited to,subcutaneous, intravenous, intramuscular, intrasternal, infusiontechniques, intraperitoneal administration and parenteraladministration.

II. Fusion Protein A. General

The fusion protein is constructed to include a ligand binding domain anda nucleic acid binding domain; the nucleic acid binding domain is notderived from the same receptor as the ligand binding domain. Inclusionof these two domains permits sequence specific binding to target nucleicacid sequences present in endogenous or exogenous nucleic acidmolecules. It also provides ligand-dependent control of suchsequence-specific binding. The fusion protein can also include atranscription regulating domain that serves to enhance, suppress oractivate expression of an endogenous or exogenous gene. Suchtranscriptional control is also ligand dependent.

The nucleic acid binding domain (the DBD) includes one or more zincfinger peptide modular units, and typically a plurality of such unitsjoined to provide a peptide designed to bind to the regulatory region ina targeted gene. Zinc fingers provide a means to design DBDs of adesired specificity.

The fusion protein also includes a LBD that is derived from anintracellular receptor, preferably a hormone receptor, more preferably asteroid receptor. The LBD can be modified to have altered ligandspecificity so that endogenous or natural ligands do not interact withit, but non-natural ligands do. The fusion protein also can include atranscription regulating domain (TRD) that regulates transcription ofthe targeted gene(s). In some embodiments, the TRD can represstranscription of an endogenous gene; in others it can activateexpression of an endogenous or exogenous gene.

Hence the fusion protein is made by operably linking a LBD domain froman intracellular receptor to one or more zinc finger domains, selectedto bind to a targeted gene. A transcription regulating domain can alsobe operably linked. This is accomplished by any method known to those ofskill in the art. Generally the fusion protein is produced by expressingnucleic acid encoding the fusion protein.

1. Ligand Binding Domain (LBD)

The ligand binding domain is derived from an intracellular receptor, andis preferably derived from a nuclear hormone receptor. The LBD of anintracellular receptor includes the approximately 300 amino acids fromthe carboxy terminus, which can be used with or without modification.

By mutation of a small number of residues ligand specificity can bealtered. The ligand binding domain can be modified, such as bytruncation or point mutation to alter its ligand specificity permittinggene regulation by non-natural or non-native ligands.

Exemplary hormone receptors are steroid receptors, which are well knownin the art. Exemplary and preferred steroid receptors include estrogenand progesterone receptors and variants thereof. Of particular interestare ligand binding domains that exhibit altered ligand specificity sothat the LBD does not respond to the natural hormone, but rather to adrug, such as RU486, or other inducer. Means to modify and test thespecificity of ligand binding domains and to identify ligands thereforare known (see, U.S. Pat. No. 5,874,534; U.S. Pat. No. 5,935,934; andInternational PCT application No. 98/18925, which is based on U.S.provisional application Ser. No. 60/029,964; International PCTapplication No. 96/40911, which is based on U.S. application Ser. No.08/479,913).

The LBD can be modified by deletion of from about 1 up to about 150,typically 120, amino acids on the carboxyl terminal end of the receptorfrom which the LBD derives. Systematic deletion of amino acids andsubsequent testing of the ligand specificity and of the resulting LBDcan be used to empirically identify mutations that lead to modified LBDsthat have desired properties, such as preferential interaction withnon-natural ligands. Exemplary mutations are described in the Examplesherein, and also are known to those of skill in the art (see, e.g., U.S.Pat. No. 5,874,534; U.S. Pat. No. 5,935,934; U.S. Pat. No. 5,364,791;and International PCT application No. 98/18925, which is based on U.S.provisional application Ser. No. 60/029,964; International PCTapplication No. 96/40911, which is based on U.S. application Ser. No.08/479,9131) and references cited therein. Hence a LBD or modified formthereof prepared by known methods is obtained and operably linked to aDBD; a TRD is also linked as needed.

2. Nucleic Acid Binding Domain (DBD)

Zinc fingers are modular nucleic acid binding peptides. The zincfingers, or modules thereof, or variants thereof can be used toconstruct fusion proteins that specifically interact with targetedsequences. Zinc fingers are ubiquitous proteins, and many arewell-characterized. For example, methods and rules for preparation andselection of zinc fingers based upon the C2H2 class of zinc fingers withunique specificity are known (see, e.g., International PCT applicationNo. WO 98/54311 and International PCT application No. 95/19431; see,also U.S. Pat. No. 5,789,538; Beerli et al. (1999) Proc. Natl. Acad.Sci. U.S.A. 96:2758-2763; Beerli et al. (1995) Proc. Natl. Acad. Sci.U.S.A. 95:14628-14633; see, also U.S. application Ser. No. 09/173,941,filed 16 October, 1998, published as International PCT application No.WO 00/23464). Exemplary targeting sequences are provided herein.

Furthermore, other zinc fingers can be similarly identified and therules known for the C2H2 can be applied to modification of thespecificity of such zinc fingers or alternative rules unique to eachclass can be deduced in a similar manner.

The advantage of using zinc fingers for targeting of theligand-dependent transcription regulating fusion proteins providedherein is the ability to construct zinc fingers with unique specificity.This permits targeting and ligand-dependent control of expression ofspecific endogenous genes and also ligand-dependent control ofexogenously administered genes, such as genes that encode therapeuticproducts.

Zinc fingers and modular units thereof can be obtained or prepared byany method known to those of skill in the art. As discussed herein, aplethora of zinc fingers, including synthetic zinc fingers having avariety of sequence specificities are known, as are means for combiningthe modular domains to produce a resulting peptide that binds to anydesired target sequence of nucleic acids. Rules for creating zincfingers of desired specificity are known and can be deduced by methodsused by those of skill in the art (see, e.g., (see, e.g., InternationalPCT application No. WO 98/54311, which is based on U.S. application Ser.No. 08/863,813; International PCT application No. 95/19431, which isbased on U.S. application Ser. Nos. 08/183,119 and 08/312,604).

For example, zinc finger variants can be prepared by identifying a zincfinger or modular unit thereof, creating an expression library, such asa phage display library (see, e.g., International PCT application No. WO98/54311, Barbas et al. (1991) Methods 2:119; Barbas et al. (1992) Proc.Natl. Acad. Sci. U.S.A. 89:4457), encoding polypeptide variants of thezinc finger or modular units thereof, expressing the library in a hostand screening for variant peptides having a desired specificity. Zincfingers may also be constructed by combining amino acids (or encodingnucleic acids) according to the known rules of binding specificity and,if necessary, testing or screening the resulting peptides to ensure thepeptide has a desired specificity. Because of the modular nature of zincfingers, where each module can be prepared to bind to a three nucleotidesequence, peptides of any specificity can be prepared from the modules.The number of modules used depends upon the specificity of genetargeting desired. Modular units are combined; spacers (i.e. TGEKP,TGQKP) required to maintain spacing and conformational features of themodular domains are included in the peptide (see, e.g., WO 98/54311).

a. Zinc Fingers as DBDs and Zinc Finger Modular Units

The nucleic acid binding domain in the fusion protein includes zincfinger modular domains and is designed to bind to a target nucleic acidsequence present in an endogenous gene or in an exogenous gene that isadministered in combination with the fusion protein or nucleic acidencoding the fusion protein.

Zinc fingers are among the most common and ubiquitous nucleic acidbinding proteins. Any zinc finger polypeptide or modular unit thereof iscontemplated; preferably the domain is non-immunogenic in the host forwhich the fusion protein is intended. For human therapy, the zinc fingerDBD preferably is selected from human zinc protein modular units orvariants thereof.

For purposes herein, the zinc finger used generally is other than thenaturally-occurring zinc finger present in the intracellular receptorfrom which the ligand binding domain is derived. Typically the fusionprotein is produced by replacing the native zinc finger present in thereceptor with the selected zinc finger designed to interact with atargeted nucleic acid regulatory region. In addition, the zinc fingerscan be designed by selection of appropriate modular units to havespecificity for a targeted gene, thereby providing a precise means tomodulate expression of a targeted gene.

Naturally occurring zinc finger proteins generally contain multiplerepeats of the zinc finger motif. This modular nature is unique amongthe different classes of DNA binding proteins. Wild type zinc fingerproteins are made up of from two to as many as 37 modular tandemrepeats, with each repeat forming a “finger” holding a zinc atom intetrahedral coordination by means of a pair of conserved cysteines and apair of conserved histidines. Generally each finger also containsconserved hydrophobic amino acids that interact to form a hydrophobiccore that helps the module maintain its shape. Polydactyl arrays of asmany as 37 zinc finger domains allow this recognition domain torecognize extended asymmetric sequences. Any such zinc finger orcombinations of modular units thereof is intended for use herein.

A zinc finger-nucleotide binding peptide domain contains a uniqueheptamer (contiguous sequence of 7 amino acid residues) within theα-helical domain of the polypeptide, which heptameric sequencedetermines binding specificity to a target nucleotide. The heptamericsequence can be located anywhere within the α-helical domain but it ispreferred that the heptamer extend from position −1 to position 6 as theresidues are conventionally numbered in the art. A peptidenucleotide-binding domain can include any β-sheet and frameworksequences known in the art to function as part of a zinc finger protein.

Studies of natural zinc finger proteins have shown that three zincfinger domains can bind 9 bp of contiguous DNA sequence (Pavletich etal. (1991) Science 252:809-817; Swirnoff et al. (1995) Mol. Cell. Biol.15:2275-2287). While recognition of 9 bp of sequence is insufficient tospecify a unique site in a complex genome, proteins containing six zincfinger domains can specify 18-bp recognition (Liu et al. (1997) Proc.Natl. Acad. Sci. USA 94:5525-5530). An 18-bp address made up of modularunits is of sufficient complexity to specify a single site within allknown genomes (see, published International PCT application No. WO98/54311). Rules for constructing Zinc finger arrays that bind to aparticular DNA sequence are known (see, e.g., International PCTapplication No. WO 98/54311, which is based on U.S. application Ser. No.08/863,813; International PCT application No. 95/19431, which is basedon U.S. application Ser. Nos. 08/183,119 and 08/312,604).

Zinc finger-nucleotide binding polypeptide variants can be constructedfrom known motifs. The variants include at least two and preferably atleast about four zinc finger modules that bind to a cellular nucleotidesequence, such as DNA, RNA or both, and specifically bind to andmodulate the function of a cellular nucleotide sequence.

For purposes herein, it is not necessary that the zinc finger-nucleotidebinding motif be known in order to obtain a zinc-finger nucleotidebinding variant polypeptide. It is contemplated that zincfinger-nucleotide binding motifs can be identified in non-eukaryotic DNAor RNA, especially in the native promoters of bacteria and viruses bythe binding thereto of the modified nucleic acid binding peptides.Modified nucleic acid binding peptides should preserve the well knownstructural characteristics of the zinc finger, but differ from zincfinger proteins found in nature by their amino acid sequences andthree-dimensional structures.

A variety of zinc finger proteins are known. Among these, the Cys₂-His₂(also referred to as “C2H2”) zinc fingers are preferred for use in thefusion proteins. There are well-defined rules for C2H2 zinc fingerbinding to DNA that allow the DNA binding specificity of the fusionproteins containing the zinc fingers to be adjusted in order to reducenon-specific interactions with genes other than the targeted genes.These proteins can be selected or engineered to bind to diversesequences. Further, the sequence specificity of these proteins can bemodified to be different from their naturally occurring targets.Examples of zinc finger proteins from which a polypeptide can beproduced include TFIIIA and Zif268.

The murine Cys₂-His₂ zinc finger protein Zif268 has been used forconstruction of phage display libraries (Wu et al. (1995) Proc. Natl.Acad. Sci. U.S.A. 92:344-348). Zif268 is structurally the most wellcharacterized of the zinc-finger proteins (Pavletich, et al. (1991)Science 252:809-817; Elrod-Erickson et al. (1996) Structure 4:1171-1180;Swirnoff et al. (1995) Mol. Cell. Biol. 15:2275-2287). DNA recognitionin each of the three zinc finger domains of this protein is mediated byresidues in the N-terminus of the α-helix contacting primarily threenucleotides on a single strand of the DNA. The operator binding site forthis three finger protein is 5′-GCGTGGGCG-′3 (finger-2 subsite isunderlined). Structural studies of Zif268 and other related zincfinger-DNA complexes have shown that residues from primarily threepositions on the α-helix, −1, 3, and 6, are involved in specific basecontacts. Typically, the residue at position −1 of the α-helix contactsthe 3′ base of that finger's subsite while positions 3 and 6 contact themiddle base and the 5′ base, respectively.

b. Construction and Isolation of Zinc Finger DBD Peptides

A zinc finger-nucleotide binding polypeptide that binds to DNA, andspecifically, the zinc finger domains that bind to DNA, can beidentified by examination of the “linker” region between two zinc fingerdomains. The linker amino acid sequence TGEK(P) (SEQ ID NO: 19) istypically indicative of zinc finger domains that bind to a DNA.Therefore, one can determine whether a particular zinc finger-nucleotidebinding polypeptide preferably binds to DNA or RNA by examination of thelinker amino acids.

c. Synthetic Zinc Fingers

Synthetic zinc fingers can be assembled based upon known sequencespecificities. A large number of zinc finger-nucleotide bindingpolypeptides were made and tested for binding specificity against targetnucleotides containing a GNN triplet. The data show that a strikingconservation of all three of the primary DNA contact positions (−1, 3,and 6) was observed for virtually all the clones of a given target (see,Example 1, see, also U.S. application Ser. No. 09/173,941, filed 16Oct., 1998, published as International PCT application No. WO 00/23464).

In order to select a family of zinc finger domains recognizing the5′-GNN-3′ subset of sequences, two highly diverse zinc finger librarieswere constructed in the phage display vector pComb3H (Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; Rader et al. (1997)Curr. Opin. Biotechnol. 8:503-508). Both libraries involvedrandomization of residues within the α-helix of finger 2 of C7, avariant of Zif268 (Wu et al. (1995) Proc. Natl. Acad. Sci. U.S.A.92:344-348). Library 1 was constructed by randomization of positions −1,1, 2, 3, 5, 6 using a NNK doping strategy while library 2 wasconstructed using a VNS doping strategy with randomization of positions−2, −1, 1, 2, 3, 5, 6. The NNK doping strategy allows for all amino acidcombinations within 32 codons while VNS precludes Tyr, Phe, Cys and allstop codons in its 24 codon set. The libraries contained 4.4×10⁹ and3.5×10⁹ members, respectively, each capable of recognizing sequences ofthe 5′-GCGNNNGCG-3′ type. The size of the NNK library ensured that itcould be surveyed with 99% confidence while the VNS library was highlydiverse but somewhat incomplete. These libraries are, however,significantly larger than previously reported zinc finger libraries(International PCT application No. WO 09/54311; Choo et al. (1994) ProcNatl Acad Sci USA 91:11163-7; Greisman et al. (1997) Science275:657-661; Rebar et al. (1994) Science 263:671-673; Jamieson et al.(1994) Biochemistry 33:5689-5695; Jamieson et al. 1996) Proc. Natl.Acad. Sci. U.S.A. 93:12834-12839; Isalan et al. (1998) Biochemistry37:12026-12033; and U.S. Pat. No. 5,789,538). Seven rounds of selectionwere performed on the zinc finger displaying-phage with each of the 165′-GCGGNNGCG-3′ biotinylated hairpin DNAs targets using a solutionbinding protocol. Stringency was increased in each round by the additionof competitor DNA. Sheared herring sperm DNA was provided for selectionagainst phage that bound non-specifically to DNA. Stringent selectivepressure for sequence specificity was obtained by providing DNAs of the5′-GCGNNNGCG-3′ types as specific competitors. Excess DNA of the5′-GCGGNNGCG-3′ type was added to provide even more stringent selectionagainst binding to DNAs with single or double base changes as comparedto the biotinylated target. Phage binding to the single biotinylated DNAtarget sequence were recovered using streptavidin coated beads. In somecases the selection process was repeated. The data show that thesedomains are functionally modular and can be recombined with one anotherto create proteins capable of binding to 18-bp sequences withsubnanomolar affinity. The resulting family of zinc finger domainsdescribed herein is sufficient for the construction of 17 millionproteins that bind to the 5′-(GNN)₆-3′ family of DNA sequences.

Also impressive amino acid conservation was been observed forrecognition of the same nucleotide in different targets. For example,Asn in position 3 (Asn3) virtually always selects to recognize adeninein the middle position, whether in the context of GAG, GAA, GAT, or GAC.Gln-1 and Arg-1 were always selected to recognize adenine or guanine,respectively, in the 3′ position regardless of context. Amide side chainbased recognition of adenine by Gln or Asn is well documented instructural studies as is the Arg guanidinium side chain to guaninecontact with a 3′ or 5′ guanine (see, e.g., Elrod-Erickson et al. (1998)Structure 6:451-464).

More often, however, two or three amino acids are selected fornucleotide recognition. His3 or Lys3 (and to a lesser extent, Gly3) areselected for the recognition of a middle guanine. Ser3 and Ala3 areselected to recognize a middle thymine. Thr3, Asp3, and Glu3 areselected to recognize a middle cytosine. Asp and Glu are selected inposition −1 to recognize a 3′ cytosine, while Thr-1 and Ser-1 areselected to recognize a 3′ thymine.

Specific recognition of many nucleotides can best accomplished usingmotifs, rather than a single amino acid. For example, the bestspecification of a 3′ guanine is achieved using the combination ofArg-1, Ser1, and Asp2 (the RSD motif). By using Val5 and Arg6 to specifya 5′ guanine, recognition of subsites GGG, GAG, GTG, and GCG can beaccomplished using a common helix structure (SRSD-X-LVR) differing onlyin the position 3 residue (Lys3 for GGG, Asn3 for GAG, Glu3 for GTG, andAsp3 for GCG). Similarly, 3′ thymine is specified using Thr-1, Ser1, andGly2 in the final clones (the TSG motif). Further, a 3′ cytosine can bespecified using Asp-1, Pro 1, and Gly2 (the DPG motif) except when thesubsite is GCC; Pro1 is not tolerated by this subsite. Specification ofa 3′ adenine is with Gln-1, Ser1, Ser2 in two clones (QSS motif).

The data (see, Table 1 in Example) show that all possible GNN tripletsequences can be recognized with exquisite specificity by zinc fingerdomains. Optimized zinc finger domains can discriminate single basedifferences by greater than 100-fold loss in affinity. While many of theamino acids found in the optimized proteins at the key contact positions−1, 3, and 6 are those that are consistent with a simple code ofrecognition, it has been discovered that optimal specific recognition issensitive to the context in which these residues are presented. Residuesat positions 1, 2, and 5 have been found to be critical for specificrecognition.

Further the data demonstrate that sequence motifs at positions −1, 1,and 2 rather than the simple identity of the position 1 residue arerequired for highly specific recognition of the 3′ base. These residueslikely provide the proper stereo-chemical context for interactions ofthe helix in terms of recognition of specific bases and in the exclusionof other bases, the net result being highly specific interactions. Readyrecombination of the disclosed domains then allows for the creation ofproteins, typically polydactyl proteins, of defined specificityprecluding the need to develop phage display libraries in theirgeneration. Such family of zinc finger domains is sufficient for theconstruction of 16 or 17 million proteins that bind to the 5′-(GNN)₆-3′family of DNA sequences.

d. Modification of Zinc Finger Peptides

The zinc finger-nucleotide binding peptide domain can be derived orproduced from a wild type zinc finger protein by truncation orexpansion, or as a variant of the wild type-derived polypeptide by aprocess of site directed mutagenesis, or by a combination of theprocedures (see, e.g., U.S. Pat. No. 5,789,538, which describes methodsfor design and construction of zinc finger peptides). Mutagenesis can beperformed to replace non-conserved residues in one or more of therepeats of the consensus sequence. Truncated zinc finger-nucleotidebinding proteins can also be mutagenized.

DNA encoding the zinc finger-nucleotide binding proteins, includingnative, truncated, and expanded polypeptides, can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationprocedures which are well known in the art. These include, but are notlimited to: (1) hybridization of probes to genomic or cDNA libraries todetect shared nucleotide sequences; (2) antibody screening of expressionlibraries to detect shared structural features; and (3) synthesis by thepolymerase chain reaction (PCR). RNA can be obtained by methods known inthe art (seem e.g., Current Protocols in Molecular Biology, 1988, Ed.Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience).

DNA encoding zinc finger-nucleotide binding proteins also can beobtained by: (1) isolation of a double-stranded DNA sequence from thegenomic DNA; (2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and (3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods the isolation ofgenomic DNA is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

For obtaining zinc finger derived-DNA binding polypeptides, thesynthesis of DNA sequences is frequently the method of choice when theentire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the formation of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucleic Acid Research, 11:2325,1983).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. By using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981; Maniatis, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, 1982).

Screening procedures that rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA.

A cDNA expression library, such as lambda gt11, can be screenedindirectly for zinc finger-nucleotide binding protein or for the zincfinger derived polypeptide having at least one epitope, using antibodiesspecific for the zinc finger-nucleotide binding protein. Such antibodiescan be either polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of zinc finger-nucleotidebinding protein cDNA. Alternatively, binding of the derived polypeptidesto DNA targets can be assayed by incorporated radiolabeled DNA into thetarget site and testing for retardation of electrophoretic mobility ascompared with unbound target site.

A preferred vector used for identification of truncated and/ormutagenized zinc finger-nucleotide binding polypeptides is a recombinantDNA molecule containing a nucleotide sequence that codes for and iscapable of expressing a fusion polypeptide containing, in the directionof amino- to carboxy-terminus, (1) a prokaryotic secretion signaldomain, (2) a heterologous polypeptide, and (3) a filamentous phagemembrane anchor domain. The vector includes DNA expression controlsequences for expressing the fusion polypeptide, preferably prokaryoticcontrol sequences.

Since the DNA sequences provided herein encode essentially all or partof an zinc finger-nucleotide binding protein, it is routine to prepare,subclone, and express the truncated polypeptide fragments of DNA fromthis or corresponding DNA sequences. Alternatively, by using the DNAfragments disclosed herein, which define the zinc finger-nucleotidebinding polypeptides, it is possible, in conjunction with knowntechniques, to determine the DNA sequences encoding the entire zincfinger-nucleotide binding protein. Such techniques are described in U.S.Pat. No. 4,394,443 and U.S. Pat. No. 4,446,235, which are incorporatedherein by reference.

In addition to modifications in the amino acids making up the zincfinger, the zinc finger derived polypeptide can contain more or lessthan the full amount of fingers contained in the wild type protein fromwhich it is derived. Minor modifications of the primary amino acidsequence may result in proteins which have substantially equivalentactivity compared to the zinc finger derived-binding protein describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All proteins produced by thesemodifications are included herein as long as zinc finger-nucleotidebinding protein activity exists.

e. Screening of Variant Zinc Finger and Other DBD Peptides

Any method known to those of skill in the art for identification offunctional modular domains derived from zinc fingers and combinationsthereof can be employed. An exemplary method for identifying variants ofzinc fingers or other polypeptides that bind to zinc finger bindingmotifs is provided. Components used in the method include a nucleic acidmolecule encoding a putative or modified zinc finger peptide operablylinked to a first inducible promoter and a reporter gene operably linkedto a second inducible promoter and a zinc finger-nucleotide bindingmotif, wherein the incubating is carried out under conditions sufficientto allow the components to interact, and measuring the affect of theputative DBD peptide on the expression of the reporter gene is provided.

For example, a first inducible promoter, such as the arabinose promoter,is operably linked to the nucleotide sequence encoding the putative DBDpolypeptide. A second inducible promoter, such as the lactose promoter,is operably linked to a zinc finger derived-DNA binding motif followedby a reporter gene, such as β-galactosidase. Incubation of thecomponents may be in vitro or in vivo. In vivo incubation may includeprokaryotic or eukaryotic systems, such as E. coli or COS cells,respectively. Conditions that allow the assay to proceed includeincubation in the presence of a substance, such as arabinose andlactose, which activate the first and second inducible promoters,respectively, thereby allowing expression of the nucleotide sequenceencoding the putative trans-modulating protein nucleotide sequence.Determination of whether the putative modulating protein binds to thezinc finger-nucleotide binding motif, which is operably linked to thesecond inducible promoter, and affects its activity is measured by theexpression of the reporter gene. For example, if the reporter gene isβ-galactosidase, the presence of blue or white plaques indicates whetherthe putative modulating protein enhances or inhibits, respectively, geneexpression from the promoter. Other commonly used assays to assess thefunction from a promoter, including chloramphenicol acetyl transferase(CAT) assay, are known to those of skill in the art. Prokaryote andeukaryote systems can be used.

As discussed above, Example 1 provides an illustration of modificationof Zif268 as described above. Therefore, in another embodiment, a ligandactivated transcriptional regulator polypeptide variant containing atleast two zinc finger modules that bind to an HIV sequence and modulatesthe function of the HIV sequence, for example, the HIV promoter sequenceis provided.

In another embodiment, zinc finger proteins can be manipulated torecognize and bind to extended target sequences. For example, zincfinger proteins containing from about 2 to 20 zinc fingers Zif(2) toZif(20), and preferably from about 2 to 12 zinc fingers, may be fused tothe leucine zipper domains of the Jun/Fos proteins, prototypical membersof the bZIP family of proteins (O'Shea et al. (1991) Science 254:539).Alternatively, zinc finger proteins can be fused to other proteins whichare capable of forming heterodimers and contain dimerization domains.Such proteins are known to those of skill in the art.

The Jun/Fos leucine zippers are described for illustrative purposes andpreferentially form heterodimers and allow for the recognition of 12 to72 base pairs. Henceforth, Jun/Fos refer to the leucine zipper domainsof these proteins. Zinc finger proteins are fused to Jun, andindependently to Fos by methods commonly used in the art to linkproteins. Following purification, the Zif-Jun and Zif-Fos constructs,the proteins are mixed to spontaneously form a Zif-Jun/Zif-Fosheterodimer. Alternatively, coexpression of the genes encoding theseproteins results in the formation of Zif-Jun/Zif-Fos heterodimers invivo. Fusion of the heterodimer with an N-terminal nuclear localizationsignal allows for targeting of expression to the nucleus (Kalderon, etal, Cell, 39:499-509, 1984). Activation domains may also be incorporatedinto one or each of the leucine zipper fusion constructs to produceactivators of transcription (Sadowski et al. (1992) Gene 118:137). Thesedimeric constructs then allow for specific activation or repression oftranscription. These heterodimeric Zif constructs are advantageous sincethey allow for recognition of palindromic sequences (if the fingers onJun and Fos recognize the same DNA/RNA sequence) or extended asymmetricsequences (if the fingers on Jun and Fos recognize different DNA/RNAsequences). For example the palindromic sequence

5′-GGC CCA CGC {N}_(X) GCG TGG GCG-3′ 3′-GCG GGT GCG {N}_(X) CGC ACCCGC-5′ (SEQ ID NO: 20)is recognized by the Zif268-Fos/Zif268 Jun dimer (x is any number). Thespacing between subsites is determined by the site of fusion of Zif withthe Jun or Fos zipper domains and the length of the linker between theZif and zipper domains. Subsite spacing is determined by a binding siteselection method as is common to those skilled in the art (Thiesen etal. (1990) Nucleic Acids Research, 18:3203, 1990). Example of therecognition of an extended asymmetric sequence is shown by theZif(C7)₆-Jun/Zif-268-Fos dimer. This protein includes 6 fingers of theC7 type (EXAMPLE 11) linked to Jun and three fingers of Zif268 linked toFos, and recognizes the extended sequence:

(SEQ ID NO: 21) 5′-CGC CGC CGC CGC CGC CGC {N}_(X) GCG TGG GCG-3′ 3′-GCGGCG GCG GCG GCG GCG {N}_(X) CGC ACC CGC-5′

In another embodiment, attachment of chelating groups to Zif proteins ispreferably facilitated by the incorporation of a Cysteine (Cys) residuebetween the initial Methionine (Met) and the first Tyrosine (Tyr) of theprotein. The Cys is then alkylated with chelators known to those skilledin the art, for example, EDTA derivatives as described (Sigman (1990)Biochemistry, 29:9097). Alternatively the sequence Gly-Gly-His can bemade as the most amino terminal residues since an amino terminuscomposed of the residues has been described to chelate Cu⁺² (Mack et al.(1988) J. Am. Chem. Soc. 110:7572). Preferred metal ions include Cu⁺²,Ce⁺³ (Takasaki and Chin (1994) J. Am. Chem. Soc. 116:1121, 1994) Zn⁺²,Cd⁺², Pb⁺², Fe⁺² (Schnaith et al. (1994) Proc. Natl. Acad. Sci., USA91:569, 1994), Fe⁺³, Ni⁺², Ni⁺³, La⁺³, Eu⁺³ (Hall et al. (1994)Chemistry and Biology 1:185), Gd⁺³, Tb⁺³, Lu⁺³, Mn⁺², Mg⁺². Cleavagewith chelated metals is generally performed in the presence of oxidizingagents such as O₂, hydrogen peroxide H₂O₂ and reducing agents such asthiols and ascorbate. The site and strand (+ or − site) of cleavage isdetermined empirically (Mack et al. (1988) J. Am. Chem. Soc 110:7572,1988) and is dependent on the position of the Cys between the Met andthe Tyr preceding the first finger. In the protein Met (AA)Tyr-(Zif)₁₋₁₂, the chelate becomes Met-(AA)_(x1)Cys-Chelate-(AA)_(x2)-Tyr-(Zif)₁₋₁₂, where AA=any amino acid and x=thenumber of amino acids. Dimeric zif constructs of the typeZif-Jun/Zif-Fos are preferred for cleavage at two sites within thetarget oligonucleotide or at a single long target site. In the casewhere double stranded cleavage is desired, Jun and Fos containingproteins are labelled with chelators and cleavage is performed bymethods known to those skilled in the art. In this case, a staggereddouble-stranded cut analogous to that produced by restriction enzymes isgenerated.

Following mutagenesis and selection of variants of the Zif268 protein inwhich the finger 1 specificity or affinity is modified, proteinscarrying multiple copies of the finger may be constructed using theTGEKP linker sequence by methods known in the art. For example, the C7finger may be constructed according to the scheme:MKLLEPYACPVESCDRRFSKSADLKRHIRHTGEKP- (SEQ ID NO: 22)(YACPVESCDRRFSKSADLKHIRIHTGEKP)₁₋₁₁, (SEQ ID NO: 23) where the sequenceof the last linker is subject to change since it is at the terminus andnot involved in linking two fingers together. This protein binds thedesigned target sequence GCG-GCG-GCG in the oligonucleotide hairpinCCT-CGC-CGC-CGC-GGG-TTT-TCC-CGC-GCC-CCC GAG G (SEQ ID NO: 24) with anaffinity of 9 nM, as compared to an affinity of 300 nM for anoligonucleotide encoding the GCG-TGG-GCG sequence (as determined bysurface plasmon resonance studies). Fingers used need not be identicaland may be mixed and matched to produce proteins which recognize adesired target sequence. These may also be used with leucine zippers(e.g., Fos/Jun) or other heterodimers to produce proteins with extendedsequence recognition.

In addition to producing polymers of finger 1, the entire three fingerZif268 and modified versions therein may be fused using the consensuslinker TGEKP to produce proteins with extended recognition sites. Forexample, the protein Zif268-Zif268 can be produced in which the naturalprotein has been fused to itself using the TGEKP linker. This proteinnow binds the sequence GCG-TGG-GCG-GCG-TGG-GCG. Therefore modificationswithin the three fingers of Zif268 or other zinc finger proteins knownin the art may be fused together to form a protein which recognizesextended sequences. These new zinc proteins may also be used incombination with leucine zippers if desired.

3. Transcription Regulating Domain (TRD)

Any TRD known to those of skill in the art can be selected, includingthose present in intracellular receptors. The TRD is selected toregulate transcription of the gene targeted by the DBD and to effectregulation of expression thereof. The TRD can be selected to regulateexpression of an endogenous gene in a cell or in an exogenously addedconstruct. For exogenously added genes, the regulatory region of thegene can be selected to interact with a desired TRD. Identification,preparation and testing of TRDs in combination with DBDs is exemplifiedherein for ERB-2 and integrin β₃.

a. Selection of the TRD

Transcription regulating domains are well known in the art. Exemplaryand preferred transcription repressor domains are ERD, KRAB, SID,Deacetylase, and derivatives, multimers and combinations thereof such asKRAB-ERD, SID-ERD, (KRAB)₂, (KRAB)₃, KRAB-A, (KRAB-A)₂, (SID)₂(KRAB-A)-SID and SID-(KRAB-A).

b. Repressors

Transcriptional repressors are well known in the art, and any suchrepressor can be used herein. The repressor is a polypeptide that isoperatively linked to the nucleic acid binding domain as set forthabove. The repressor is operatively linked to the binding domain in thatit is attached to the binding domain in such a manner that, when boundto a target nucleotide via that binding domain, the repressor acts toinhibit or prevent transcription. The repressor domain can be linked tothe binding domain using any linking procedure well known in the art. Itmay be necessary to include a linker moiety between the two domains.Such a linker moiety is typically a short sequence of amino acidresidues that provides spacing between the domains. So long as thelinker does not interfere with any of the functions of the binding orrepressor domains, any sequence can be used.

Transcriptional repressors have been generated by attaching either ofthree human-derived repressor domains to the zinc finger protein. Thefirst repressor protein was prepared using the ERF repressor domain(ERD) (Sgouras et al. (1995) EMBO J. 14:4781-4793), defined by aminoacids 473 to 530 of the ets2 repressor factor (ERF). This domainmediates the antagonistic effect of ERF on the activity of transcriptionfactors of the ets family. A synthetic repressor was constructed byfusion of this domain to the C-terminus of the zinc finger protein.

The second repressor protein was prepared using the Krüppel-associatedbox (KRAB) domain (Margolin et al. (1994) Proc. Natl. Acad. Sci. USA91:4509-4513). This repressor domain is commonly found at the N-terminusof zinc finger proteins and presumably exerts its repressive activity onTATA-dependent transcription in a distance- and orientation-independentmanner (Pengue et al. (1996) Proc. Natl. Acad. Sci. USA 93:1015-1020),by interacting with the RING finger protein KAP-1 (Friedman et al.(1996) Genes & Dev. 10:2067-2078). The KRAB domain found between aminoacids 1 and 97 of the zinc finger protein KOX1 (Margolin et al. (1994)Proc. Natl. Acad. Sci. USA 91:4509-4513) was used. In this case anN-terminal fusion with the six-finger protein was constructed.

Histone deacetylation as a means for repression can be employed. Forexample, amino acids 1 to 36 of the Mad mSIN3 interaction domain (SID)have been fused to the N-terminus of a zinc finger protein (Ayer et al.(1996) Mol. Cell. Biol. 16:5772-5781). This small domain is found at theN-terminus of the transcription factor Mad and is responsible formediating its transcriptional repression by interacting with mSIN3,which in turn interacts the co-repressor N-CoR and with the histonedeacetylase mRPD1 (Heinzel et al. (1997) Nature 387:43-46).

c. Activators

Exemplary and preferred transcription activation domains include anyprotein or factor that regulates transcription. Exemplarytranscriptional regulation domains include, but are not limited to,VP16, TA2, VP64, STAT6 and relA.

4. Exemplary Construct Based on Human Integrin P3 and erbB-2 TargetSequences

To exemplify the generation of zinc finger modular domains and peptidescontaining one or more of such domains to produce peptides with DNAbinding specificity and therapeutic potential, target sequences havebeen identified based on human integrin P3 and erbB-2 (Ishii et al.(1987) Proc. Natl. Acad. Sci. U.S.A. 84:4374-4378) genomic sequences.

Integrin β3 as a Target for Cancer Gene Therapy

Integrin α_(v)β₃ is the most promiscuous member of the integrin familyand has been identified as a marker of angiogenic vascular tissue. Forinstance, integrin α_(v)β₃ shows enhanced expression on blood vessels inhuman wound granulation tissue but not in normal skin. Following theinduction of angiogenesis, blood vessels show a four-fold increase inα_(v)β₃ expression compared to blood vessels not undergoing thisprocess. It has been reported that a cyclic peptide or monoclonalantibody antagonist of integrin α_(v)β₃ blocks cytokine- ortumor-induced angiogenesis on the chick chorioallantoic membrane.Therefore, inhibition of integrin α_(v)β₃ expression provides anapproach to block tumor-induced angiogenesis.

ErbB-2 Receptor Tyrosine Kinases as a Target for Cancer Gene Therapy

Members of the ErbB receptor family play an important role in thedevelopment of human malignancies. In particular, ErbB-2 isover-expressed as a result of gene amplification and/or transcriptionalderegulation in a high percentage of human adenocarcinomas arising atnumerous sites, including breast, ovary, lung, stomach, and salivarygland. Increased expression of ErbB-2 per se leads to constitutiveactivation of its intrinsic tyrosine kinase. Many clinical studies haveshown that patients with tumors showing elevated expression of ErbB-2have poorer prognosis. Thus, the high occurrence of its aberrantexpression in human cancer, as well as the aggressive behavior ofover-expressing tumors, make ErbB-2 an attractive target for therapy.

Generation and construction of zinc fingers and fusion proteins targetedto erbB-2 and integrin P3 are described in the EXAMPLES.

B. Regulatable Cassette

In embodiments in which the targeted gene is an exogenous gene,particularly a gene that encodes a therapeutic product, the gene isprovided as in an expression cassette operatively linked to a promoterand regulatory region with which the fusion protein specificallyinteracts. The cassette includes at least one polynucleotide domainrecognized by the corresponding zinc finger domain present in the fusionprotein and a suitable promoter to direct transcription of the exogenousgene. Typically, the regulatable expression cassette contains three tosix response elements and interacts with nucleic acid binding domain ofthe ligand activated transcriptional regulatory fusion protein.

Typically the exogenous gene encodes a therapeutic product, such as agrowth factor, that can supplement peptides, polypeptides or proteinsencoded by endogenous expressed genes, thereby providing an effectivetherapy. In several embodiments the gene encodes a suitable reportermolecule that can be detected by suitable direct or indirect means. Thecassette can be inserted into a suitable delivery vehicle forintroduction into cells. Such vehicles include, but are not limited to,human adenovirus vectors, adeno-associated vectors, murine or lentivirus derived retroviral vectors, and a variety of non-viralcompositions including liposomes, polymers, and other DNA containingconjugates.

C. Use of the Fusion Proteins for Gene Regulation

1. Delivery of the Nucleic Acids

There are available to one skilled in the art multiple viral andnon-viral methods suitable for introduction of a nucleic acid moleculeinto a target cell. Genetic modification of a cell may be accomplishedusing one or more techniques well known in the gene therapy field (HumanGene Therapy, April 1994, Vol. 5, p. 543-563; Mulligan, R. C. 1993).

The ability to regulate transgene expression, as defined in the examplesherein, can be applied to a wide variety of applications for genetherapy. The ability to control expression of an exogenously introducedtransgene is important for the safety and efficacy of most or allenvisioned cell and gene therapies. Control of transgene expression canbe used to accomplish regulation of a therapeutic protein level,ablation of a desired cell population, either the vector containingcells or others, or activation of a recombinase or other functionresulting in control of vector function within the transduced cells.Further, such control permits termination of a gene therapy treatment ifnecessary.

A number of vector systems useful for gene therapy have been describedpreviously in this application. Vectors for gene therapy include anyknown to those of skill in the art, and include any vectors derived fromanimal viruses and artificial chromosomes. The vectors may be designedfor integration into the host cell's chromosomes or to remain asextrachromosomal elements. Such vectors include, but are not limited tohuman adenovirus vectors, adeno-associated viral vectors, retroviralvectors, such as murine retroviral vectors and lentivirus-derivedretroviral vectors. Also contemplated herein are any of the variety ofnon-viral compositions for targeting and/or delivery of geneticmaterial, including, but are not limited to, liposomes, polymers, andother DNA containing compositions, and targeted conjugates, such asnucleic acids linked to antibodies and growth factors. Any deliverysystem is intended for use of delivery of the nucleic acid constructsencoding the fusion polypeptide and also targeted exogenous genes. Suchvector systems can be used to deliver the ZFP-LBD fusion proteins andthe inducible transgene cassette either in vitro or in vivo, dependingon the vector system. With adenovirus, for instance, vectors can beadministered intravenously to transduce the liver and other organs,introduced directly into the lung, or into vascular compartmentstemporarily localized by ligation or other methods. Methods forconstructing such vectors, and methods and uses thereof are known tothose skilled in the field of gene therapy.

In one embodiment, one vector encodes the fusion protein regulator and asecond vector encodes the inducible transgene cassette. Vectors can bemixed or delivered sequentially to incorporate into cells the regulatorand transgene at the appropriate amounts. Subsequent administration ofand effective amount of the ligand by standard routes would result inactivation of the transgene.

In another embodiment, the nucleic acid encoding the fusion protein andthe inducible transgene can be included in the same vector construction.In this instance, the nucleic acid encoding the fusion protein would bepositioned within the vector and expressed from a promoter in such a waythat it did not interfere with the basal expression and inducibility ofthe transgene cassette. Further, the use of cell or tissue specificpromoters to express the fusion protein confers an additional level ofspecificity on the system. Dual component vectors and use for genetherapy are known (see, e.g., Burcin et al. (1999) Proc. Natl. Acad.Sci. USA 96: 335-360, which describes an adenovirus vector fully deletedof viral backbone genes).

In another embodiment, gene therapy can be accomplished using acombination of the vectors described above. For example, a retroviralvector can deliver a stably integrated, inducible transgene cassetteinto a population of cells either in vitro (ex vivo) or in vivo.Subsequently, the integrated transgene can be activated by transducingthis same cell population with a second vector, such as an adenovirusvector capable of expressing the fusion protein, followed by theadministration of the specific ligand inducing agent. This isparticularly useful where “one time” activation of the transgene isdesired, for example as a cellular suicide mechanism. An example of thisapplication is the stable integration of an inducible transgene cassettecontaining the herpes simplex virus thymidine kinase gene (HSV Tk).Subsequent activation of this gene confers sensitivity to ganciclovirand allows ablation of this modified cell.

a. Viral Delivery Systems

Viral transduction methods for delivering nucleic acid constructs tocells are contemplated herein. Suitable DNA viral vectors for use hereinincludes, but are not limited to an adenovirus (Ad), adeno-associatedvirus (AAV), herpes virus, vaccinia virus or a polio virus. A suitableRNA virus for use herein includes but is not limited to a retrovirus orSindbis virus. It is to be understood by those skilled in the art thatseveral such DNA and RNA viruses exist that may be suitable for useherein. Adenoviral vectors have proven especially useful for genetransfer into eukaryotic cells and are widely available to one skilledin the art and is suitable for use herein.

Adeno-associated virus (AAV) has recently been introduced as a genetransfer system with potential applications in gene therapy. Wild-typeAAV demonstrates high-level infectivity, broad host range andspecificity in integrating into the host cell genome. Herpes simplexvirus type-1 (HSV-1) vectors are available and are especially useful inthe nervous system because of its neurotropic property. Vacciniaviruses, of the poxvirus family, have also been developed as expressionvectors. Each of the above-described vectors is widely available and issuitable for use herein.

Retroviral vectors are capable of infecting a large percentage of thetarget cells and integrating into the cell genome. Preferredretroviruses include lentiviruses, such as but are not limited to, HIV,BIV and SIV.

Various viral vectors that can be used for gene therapy as taught hereininclude adenovirus, herpes virus, vaccinia, adeno-associated virus(AAV), or, preferably, an RNA virus such as a retrovirus. Preferably,the retroviral vector is a derivative of a murine or avian retrovirus,or is a lentiviral vector. The preferred retroviral vector is alentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector is made target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a protein. Preferred targeting is accomplished by using anantibody to target the retroviral vector. Those of skill in the art knowof, or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the zinc finger-nucleotide binding protein polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

b. Nonviral Delivery Systems

“Non-viral” delivery techniques for gene therapy include DNA-ligandcomplexes, adenovirus-ligand-DNA complexes, direct injection of DNA,CaPO₄ precipitation, gene gun techniques, electroporation, liposomes andlipofection. Any of these methods are available to one skilled in theart and would be suitable for use herein. Other suitable methods areavailable to one skilled in the art, and it is to be understood that themethods herein may be accomplished using any of the available methods oftransfection.

Another targeted delivery system is a colloidal dispersion system.Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes, whichare preferred. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. It has been shown thatlarge unilamellar vesicles (LUV), which range in size from 0.2-4.0 μmcan encapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).

Lipofection may be accomplished by encapsulating an isolated nucleicacid molecule within a liposomal particle and contacting the liposomalparticle with the cell membrane of the target cell. Liposomes areself-assembling, colloidal particles in which a lipid bilayer, composedof amphiphilic molecules such as phosphatidyl serine or phosphatidylcholine, encapsulates a portion of the surrounding media such that thelipid bilayer surrounds a hydrophilic interior. Unilammellar ormultilammellar liposomes can be constructed such that the interiorcontains a desired chemical, drug, or, as provide herein, an isolatednucleic acid molecule.

Liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells as well as mammalian cells. In order for a liposometo be an efficient gene transfer vehicle, characteristics among thefollowing should be present: (1) encapsulation of the genes of interestat high efficiency while not compromising their biological activity; (2)preferential and substantial binding to a target cell in comparison tonon-target cells; (3) delivery of the aqueous contents of the vesicle tothe target cell cytoplasm at high efficiency; and (4) accurate andeffective expression of genetic information (Mannino, et al.,Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting uses the naturaltendency of liposomes to distribute to cells of the reticulo-endothelialsystem (RES) in organs which contain sinusoidal capillaries. Activetargeting, on the other hand, involves alteration of the liposome bycoupling the liposome to a specific ligand such as a monoclonalantibody, sugar, glycolipid, or protein, or by changing the compositionor size of the liposome in order to achieve targeting to organs and celltypes other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem are ligands and receptors permitting the targeted delivery systemto find and “home in” on the desired cells. A ligand may be any compoundof interest that interacts with another compound, such as a receptor.

In general, surface membrane proteins that bind to specific effectormolecules are referred to as receptors. Antibodies are preferredreceptors. Antibodies can be used to target liposomes to specificcell-surface ligands. For example, certain antigens expressedspecifically on tumor cells, referred to as tumor-associated antigens(TAAs), may be exploited for the purpose of targeting antibody-zincfinger-nucleotide binding protein-containing liposomes directly to themalignant tumor. Since the zinc finger-nucleotide binding protein geneproduct may be indiscriminate with respect to cell type in its action, atargeted delivery system offers a significant improvement over randomlyinjecting non-specific liposomes. A number of procedures can be used tocovalently attach either polyclonal or monoclonal antibodies to aliposome bilayer. Antibody-targeted liposomes can include monoclonal orpolyclonal antibodies or fragments thereof such as Fab, or F(ab′)₂, aslong as they bind efficiently to an the antigenic epitope on the targetcells. Liposomes may also be targeted to cells expressing receptors forhormones or other serum factors.

2. Administration

a. Delivery of Constructs to Cells

The cells may be transfected in vivo, ex vivo or in vitro. The cells maybe transfected as primary cells isolated from a patient or a cell linederived from primary cells, and are not necessarily autologous to thepatient to whom the cells are ultimately administered. Following ex vivoor in vitro transfection, the cells may be implanted into a host.Genetic modification of the cells may be accomplished using one or moretechniques well known in the gene therapy field (see, e.g., (1994) HumanGene Therapy 5:543-563).

Administration of nucleic acid molecules provided herein to a targetcell in vivo may be accomplished using any of a variety of techniqueswell known to those skilled in the art. The vectors of the methodsherein may be administered orally, parentally, by inhalation spray,rectally, or topically in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. Suppositories for rectal administration of the drug can beprepared by mixing the drug with a suitable non-irritating excipientsuch as cocoa butter and polyethylene glycols that are solid at ordinarytemperatures but liquid at the rectal temperature and therefore melt inthe rectum and release the drug.

The dosage regimen for treating a disorder or a disease with the vectorsand/or compositions provided is based on a variety of factors, includingthe type of disease, the age, weight, sex, medical condition of thepatient, the severity of the condition, the route of administration, andthe particular compound employed. Thus, the dosage regimen may varywidely, but can be determined empirically using standard methods.

The pharmaceutically active compounds (i.e., vectors) can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals. For oral administration, the pharmaceutical composition may bein the form of, for example, a capsule, a tablet, a suspension, orliquid. The pharmaceutical composition is preferably made in the form ofa dosage unit containing a given amount of DNA or viral vector particles(collectively referred to as “vector”). For example, these may containan amount of vector from about 10³-10¹⁵ viral vector particles,preferably from about 10⁶-10¹² viral particles. A suitable daily dosefor a human or other mammal may vary widely depending on the conditionof the patient and other factors, but, once again, can be determinedusing routine methods. The vector may also be administered by injectionas a composition with suitable carriers including saline, dextrose, orwater.

While the nucleic acids and/or vectors herein can be administered as thesole active pharmaceutical agent, they can also be used in combinationwith one or more vectors or other agents. When administered as acombination, the therapeutic agents can be formulated as separatecompositions that are given at the same time or different times, or thetherapeutic agents can be given as a single composition.

b. Deliver Ligand

Ligands similarly may be delivered by any suitable mode ofadministration, including by oral, parenteral, intravenous,intramuscular and other known routes. Any known pharmaceuticalformulations is contemplated.

3. Ligands

As noted, the ligands may be naturally-occurring ligands, but arepreferentially non-natural ligands with which the LBD is modified tospecifically interact. Methods for modifying the LBD are known, as aremethods for screening for such ligands.

Ligands include, non-natural ligands, hormones, anti-hormones, synthetichormones, and other such compounds. Examples of non-natural ligands,anti-hormones and non-native ligands include, but are not limited to,the following:11β-4-dimethylaminophenyl)-17α-hydroxy-17α-propinyl-4,9-estradiene-3-one(RU38486 or Mifepestone);11β-(4-dimethylaminophenyl)-17α-hydroxy-17β-(3-hydroxypropyl)-13α-methyl-4,9-gonadiene-3-one(ZK98299 or Onapristone);11β-(4-acetylphenyl)-17β-hydroxy-17α-(1-propinyl)-4,9-estradiene-3-one(ZK112993); 11β-(4-dimethylaminophenyl)-17β-hydroxy-17α-(3-hydroxy-1(Z)-propenyl-estra-4,9-diene-3-one (ZK98734);(7β11β,17β)-11-(4-dimethylaminophenyl)-7-methyl-4′,5′-dihydrospiroy′ester-4,9-diene-17,2′(3′H)-furan!-3-one (Org31806);(11β,14β,17α)-4′,5′-dihydro-11-(4-dimethylaminophenyl)y′spiroestra-4,9-diene-17,2′(3′H)-furan!-3-one (Org31376);5-alpha-pregnane-3,2-dione. Additional non-natural ligands include, ingeneral, synthetic non-steroidal estrogenic or anti-estrogeniccompounds, broadly defined as selective estrogen receptor modulators(SERMS). Exemplary compounds include, but are not limited to, tamoxifenand raloxifen.

4. Pharmaceutical Compositions and Combinations

Also provided is a pharmaceutical composition containing atherapeutically effective amount of the fusion protein, or a nucleicacid molecule encoding the fusion protein in a pharmaceuticallyacceptable carrier. Pharmaceutical compositions containing one or morefusion proteins with different zinc finger-nucleotide binding domainsare contemplated. Also provided are pharmaceutical compositionscontaining the expression cassettes, and also compositions containingthe ligands. Combinations containing a plurality of compositions arealso provided.

Preparation of the Compositions

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well known. Typically suchcompositions are prepared as sterile injectables either as liquidsolutions or suspensions, aqueous or non-aqueous, however, solid formssuitable for solution, or suspensions, in liquid prior to use can alsobe prepared. The preparation can also be emulsified. Tablets and othersolid forms are contemplated.

The active ingredient can be mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, as well as pHbuffering agents and the like which enhance the effectiveness of theactive ingredient.

The therapeutic pharmaceutical composition can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and others.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, organic esters such asethyl oleate, and water-oil emulsions.

D. Methods of Gene Regulation

Methods of regulating expression of endogenous and exogenous genes areprovided. In particular, ligand-dependent methods are provided. Inpracticing the methods, a target nucleic acid molecule containing asequence that interacts with the nucleic acid binding domain of thefusion protein exposed to an effective amount of the fusion protein inthe presence of an effective binding amount of a ligand, which can beadded simultaneous with or subsequent to the fusion protein. The nucleicacid binding domain of the fusion protein binds to a portion of thetarget nucleic acid molecule and the ligand binds to the ligand bindingdomain of the fusion protein. Exposure can occur in vitro, in situ or invivo.

The amount of zinc finger derived-nucleotide binding polypeptiderequired is that amount necessary to either displace a native zincfinger-nucleotide binding protein in an existing protein/promotercomplex, or that amount necessary to compete with the native zincfinger-nucleotide binding protein to form a complex with the promoteritself. Similarly, the amount required to block a structural gene or RNAis that amount which binds to and blocks RNA polymerase from readingthrough on the gene or that amount which inhibits translation,respectively. Preferably, the method is performed intracellularly. Byfunctionally inactivating a promoter or structural gene, transcriptionor translation is suppressed. Delivery of an effective amount of theinhibitory protein for binding to or “contacting” the cellularnucleotide sequence containing the zinc finger-nucleotide bindingprotein motif, can be accomplished by one of the mechanisms describedherein, such as by retroviral vectors or liposomes, or other methodswell known in the art.

In one embodiment, a method for inhibiting or suppressing the functionof a cellular gene or regulatory sequence that includes a zincfinger-nucleotide binding motif. This is effected by contacting the zincfinger-nucleotide binding motif with an effective amount of a fusionprotein that includes zinc finger-nucleotide binding polypeptidederivative that binds to the motif. In instances in which the cellularnucleotide sequence is a promoter, the method includes inhibiting thetranscriptional transactivation of a promoter containing a zincfinger-DNA binding motif. The zinc finger-nucleotide binding polypeptidederivative may bind to a motif within a structural gene or within an RNAsequence.

Treatments

Methods for gene therapy are provided. The fusion proteins areadministered either as a protein or as a nucleic acid encoding theprotein and delivered to cells or tissues in a mammal, such as a human.The fusion protein is targeted either to a specific sequence in thegenome (an endogenous gene) or to an exogenously added gene, which isadministered as part of an expression cassette. Prior to, simultaneouswith or subsequent to administration of the fusion protein, a ligandthat specifically interacts with the LBD in the fusion protein isadministered. In embodiments, in which the targeted gene is exogenous,the expression cassette, which can be present in a vector, isadministered, simultaneous with or subsequent to administration of thefusion protein. These methods are intended for treatment of any geneticdisease, for treatment of acquired disease and any other conditions.Diseases include, cell proliferative disorders, such as cancer. Suchtherapy achieves its therapeutic effect by introduction of the fusionprotein that includes the zinc finger-nucleotide binding polypeptide,either as the fusion protein or encoded by a nucleic acid molecule thatis expressed in the cells, into cells of animals having the disorder.Delivery of the fusion protein or nucleic acid molecule can be effectedby any method known to those of skill in the art, including methodsdescribed herein. For example, it can be effected using a recombinantexpression vector such as a chimeric virus or a colloidal dispersionsystem.

The fusion proteins provided herein can be used for treating a varietyof disorders. For example the proteins can be used for treatingmalignancies of the various organ systems, including but are not limitedto, lung, breast, lymphoid, gastrointestinal, and genito-urinary tractadenocarcinomas, and other malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer, non-small cell carcinoma of thelung, cancer of the small intestine, and cancer of the esophagus. Apolynucleotide encoding the zinc finger-nucleotide binding polypeptideis also useful in treating non-malignant cell-proliferative diseasessuch as psoriasis, pemphigus vulgaris, Behcet's syndrome, and lipidhistiocytosis. Essentially, any disorder that is etiologically linked tothe activation of a zinc finger-nucleotide binding motif containingpromoter, structural gene, or RNA, would be considered susceptible totreatment with a polynucleotide encoding a derivative or variant zincfinger derived-nucleotide binding polypeptide.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Construction and Testing of Designed Specific Zinc FingerDomains

Variant zinc finger proteins have been designed and constructed toselectively bind to specific DNA sequences (Table 1). Table 1, below,summarizes the sequences (SEQ ID NO: 77-92) showing the highestselectivity for the sixteen embodiment of GNN target triplets.

TABLE 1 Target Amino acids positions Specificity −1 1 2 3 4 5 6 SEQ IDNO: GAA Q S S N L V R 77 GAC D P G N L V R 78 GAG R S D N L V R 79 GATT S G N L V R 80 GCA Q S G D L R R 81 GCC D C R D L A R 82 GCG R S D D LV K 83 GCT T S G E L V R 84 GGA Q R A H L E R 85 GGC D P G H L V R 86GGG R S D K L V R 87 GGT T S G H L V R 88 GTA Q S S S L V R 89 GTC D P GA L V R 90 GTG R S D E L V R 91 GTT T S G S L V R 92

Oligonucleotides for Zinc Finger Library Panning

Biotinylated, hairpin-structured target site oligos for panning offinger 2 (“F2”) libraries had the following sequence:

F2XXX: (SEQ ID NO: 25) 5′-Biotin-GGA CGC N′N′N′ CGC GGG TTTT CCC GCG NNNGCG TCC-3′ where NNN = either of the 16 triplets of the GNN set, or TGAand N′N′N′ = its complement.

Non-biotinylated, hairpin structured specific competitor oligos had thefollowing sequence:

F2NNN: (SEQ ID NO: 25) 5′-GGA CGC N′N′N′ CGC GGG TTTT CCC GCG NNNGCG TCC-3′ where NNN = a mixture of all 64 existing triplets andN′N′N′ = its complement.

Panning of Zinc Finger Libraries

Panning of zinc finger phage display libraries was carried out insolution using biotinylated target site hairpin oligos. Seven rounds ofpanning were carried out as follows: Phage prepared from an overnightculture was allowed to pre-bind to varying amounts of non-biotinylatedspecific competitor hairpin oligo prior to the addition of the targetsite oligo. The pre-binding was carried out in 400 μl Zinc buffer Acontaining 1% Blotto, 5 mM DTT, 4 μg sheared herring sperm DNA and 100μl phage preparation. Typically, 10 times less specific competitor thantarget oligo was used for the first round of panning. For the subsequentpanning rounds, the amount of specific competitor was graduallyincreased, up to a maximum of 12 μg in the last panning round(s).Following 30 minutes at room temperature, 100 μl Zinc buffer Acontaining 0.4 μg biotinylated target hairpin oligo were added. After2.5 to 3.5 hours at RT, phage bound to the target oligo was collected bythe addition of 50 μl Dynabeads M-280 suspension (Dynal) and incubationfor one hour at RT. The beads were collected with a magnet, washed 10times with Zinc buffer A (10 mM Tris, pH 7.5/90 mM KCl/1 mM MgCl₂/90 μMZnCl₂) containing 2% Tween-20 and 5 mM DTT, and once with Zinc buffer Acontaining 5 mM DTT. Phage was eluted for 30 minutes at RT with 25 μl ofTBS containing 10 mg/ml trypsin. Following the addition of 75 μl SuperBroth, eluted phage was allowed to infect 5 ml of E. coli ER2537 culturefor 30 minutes in a 37 degrees Celsius shaker. The volume was increasedto 10 ml and Carbenicillin was added to a concentration of 20 μg/ml. Atthis stage, the number of output phage was determined by platingaliquots of the infected bacteria onto Carbenicillin-containing LB-agarplates. After one hour shaking at 37 degrees Celsius, the Carbenicillinconcentration was increased to 50 μg/ml. After one more hour shaking at37 degrees Celsius, 10¹³ pfu helper phage was added and the culture wasincubated for a few minutes at RT. Then, 90 ml of Super Broth containingCarbenicillin (50 μg/ml) and ZnCl₂ (90 μM) were added and the culturewas incubated at 37 degrees Celsius for two hours. Upon addition ofKanamycin to a final concentration of 70 μg/ml, the culture wasincubated in a 37 degrees Celsius shaker overnight. Phage was purifiedfrom culture supernatants by PEG precipitation and resuspended in 2 mlZinc buffer A containing 1% BSA and 5 mM DTT for further rounds ofpanning. The number of phage was determined by using various dilutionsof the phage prep for infection of E. coli ER2537, followed by platingonto Carbenicillin-containing LB-agar plates. Following seven rounds ofpanning, zinc finger cDNAs were subcloned into the bacterial expressionvector pMal-CSS, a derivative of pMal-C2 (New England Biolabs), allowingfor expression of the zinc finger proteins as maltose binding protein(MBP) fusions.

Generation of Proteins with Desired DNA Binding Specificity.

To generate DNA encoding three-finger proteins, F2 coding regions werePCR amplified from selected or designed F2 variants and assembled by PCRoverlap extension. Alternatively, DNAs encoding three-finger proteinswith a Zif268 or Sp1C framework were synthesized from 8 or 6 overlappingoligonucleotides, respectively. Sp1C framework constructs were generatedas follows.

In the case of E2C-HS1(Sp1), 0.4 pmole each of oligonucleotides SPE2-3(5′-GCG AGC AAG GTC GCG GCA GTC ACT AAA AGA TTT GCC GCA CTC TGG GCA TTTATA CGG TTT TTC ACC-3′ (SEQ ID NO: 26) and SPE2-4 (5′GTG ACT GCC GCG ACCTTG CTC GCC ATC AAC GCA CTC ATA CTG GCG AGA AGC CAT ACA AAT GTC CAG AATGTG GC-3′) (SEQ ID NO: 27) were mixed with 40 pmole each ofoligonucleotides SPE2-2 (5′-GGT AAG TCC TTC TCT CAG AGC TCT CAC CTG GTGCGC CAC CAG CGT ACC CAC ACG GGT GAA AAA CCG TAT AAA TGC CCA GAG-3′) (SEQID NO: 28) and SPE2-5 (5′-ACG CAC CAG CTT GTC AGA GCG GCT GAA AGA CTTGCC ACA TTC TGG ACA TTT GTA TGG C-3′) (SEQ ID NO:29) in a standard PCRmixture and cycled 25 times (30 seconds at 94 degrees Celsius, 30seconds at 60 degrees Celsius, 30 seconds at 72 degrees Celsius). Analiquot of this pre-assembly reaction was then amplified with 40 pmoleeach of the primers SPE2-1 (5′-GAG GAG GAG GAG GTG GCC CAG GCG GCC CTCGAG CCC GGG GAG AAG CCC TAT GCT TGT CCG GAA TGT GGT AAG TCC TTC TCT CAGAGC-3′) (SEQ ID NO: 30) and SPE2-6 (5′-GAG GAG GAG GAG CTG GCC GGC CTGGCC ACT AGT TTT TTT ACC GGT GTG AGT ACG TTG GTG ACG CAC CAG CTT GTC AGAGCG-3′) (SEQ ID NO: 31) using the same cycling conditions.

The E2C-HS2(Sp1), B3B-HS1(Sp1), B3B-HS2(Sp1), B3C2-HS1(Sp1), andB3C2-HS2(Sp1) DNAs were generated in the same way, using analogous setsof oligonucleotides differing only in the recognition helix codingregions. All assembled three-finger coding regions were digested withthe restriction endonuclease Sfi1 and cloned into pMal-CSS, a derivativeof the bacterial expression vector pMal-C2 (New England Biolabs),allowing for expression of the zinc finger proteins as MBP fusions. DNAsencoding six-finger proteins with each of the different frameworks wereassembled in pMal-CSS using Xma1 and BsrF1 restriction sites included inthe sequences flanking the three-finger coding regions (Beerli et al.(1998) Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633).

Preparation of MBP-Zinc Finger Fusion Proteins for ELISA Assays

Plasmid pMal constructs containing the zinc-finger coding sequences weretransformed into the E. coli strain XL1-Blue by electroporation. Threemilliliters of Super Broth were inoculated and grown overnight at 37degrees Celsius. The next day, the cultures were diluted 1:20 in 50 mlconical tubes and grown at 37 degrees Celsius until OD₆₀₀=0.5. IPTG wasadded to a final concentration of 0.3 mM, and incubation was continuedfor 2 hours. The cultures were centrifuged for 20 minutes, then thepellets resuspended in 400 μl of Zinc Buffer A containing 5 mM freshDTT. The samples were then frozen in dry ice/ethanol and thawed in 37degrees Celsius water 6 times, then finally centrifuged for 30 secondsand left on ice for 30 minutes before use of the supernatants.

ELISA Assays

Streptavidin at a concentration of 0.2 μg/25 μl in PBS was added to eachwell of a 96 well plate, then incubated for 1 hour at 37 degreesCelsius. The plate was washed 2× with water, then biotinylated oligo at0.1 μg/25 μl in PBS, or just PBS, was added to the appropriate wells andincubated for 1 hour at 37 degrees Celsius. The plate was washed 2× withwater, then each well was filled with 3% BSA in PBS and incubated for 1hour at 37 degrees Celsius. The BSA was removed without washing, and 25μl of the appropriate extract diluted in Zinc buffer A containing 5 mMDTT was added to the appropriate wells. The binding reaction was allowedto proceed for 1 hour at room temperature. The plate was washed 8× withwater, then α-MBP mAb in Zinc buffer A and 1% BSA was added to the wellsfollowed by incubation for 30 minutes at room temperature. The plate waswashed 8× with water, then anti-mouse mAb conjugated to alkalinephosphatase in Zinc buffer A was added, and the plate was incubated for30 minutes at room temperature. After 8 final washes with water, 25 μlof alkaline phosphatase substrate and developer was added to each well.Incubation was performed at room temperature, and the OD₄₀₅ of each wellwas determined at 30 minute and 1 hour time points.

Construction of Zinc Finger-Transcription Regulating Domain FusionProteins

cDNA encoding amino acids 473 to 530 of the ets repressor factor (ERF)repressor domain (ERD) (Sgouras et al. (1995) EMBO J. 14:4781-4793) wasgenerated from four overlapping oligonucleotides using Taq DNApolymerase; a cDNA encoding amino acids 1 to 97 of the KRAB domain ofKOX1 (Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91:4509-4513)was assembled from 6 overlapping oligonucleotides; a cDNA encoding aminoacids 1 to 36 of the Mad sin3 interaction domain (SID) (Ayer et al.(1996) Mol. Cell. Biol. 16:5772-5781) was assembled from 3 overlappingoligonucleotides. The coding region for amino acids 413 to 489 of theVP16 transcriptional activation domain (Sadowski et al., (1988) Nature335:563-564) was PCR amplified from pcDNA3/C7-C7-VP16 (Liu et al. (1997)Proc. Natl. Acad. Sci. U.S.A. 94:5525-5530). The VP64 DNA, encoding atetrameric repeat of VP 16's minimal activation domain, comprising aminoacids 437 to 447 (Seipel et al. (1992) EMBO 13:4961), was generated fromtwo pairs of complementary oligonucleotides. All resulting effectordomain-encoding fragments were fused to zinc finger coding regions bystandard cloning procedures, such that each resulting constructcontained an internal SV40 nuclear localization signal, as well as aC-terminal HA decapeptide tag. Fusion constructs were cloned into pcDNA3for expression in mammalian cells.

Construction of Integrin β3 and erbB-2 Luciferase Reporter Plasmids

An integrin β3 promoter fragment encompassing nucleotides −584 to −1(with respect to the ATG codon) was PCR amplified from human genomicDNA, using the primers b3p(Nhe1)-f (5′-GAG GAG GAG GCT AGC GGG ATG TGGTCT TGC CCT CAA CAG GTA GG-3′) (SEQ ID NO: 32) and b3p(Hind3)-b (5′-GAGGAG GAG AAG CTT CTC GTC CGC CTC CCG CGG CGC TCC GC-3′) (SEQ ID NO: 33),and Taq Expand DNA Polymerase mix (Boehringer). The cycling conditionswere: 30 minutes at 94 degrees Celsius; 40× (one minute at 94 degreesCelsius-30 minutes at 62 degrees Celsius-2.5 minutes at 72 degreesCelsius); 10 minutes at 72 degrees Celsius. 10% DMSO was present in thereaction mix.

An erbB-2 promoter fragment (Ishii et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:4374-4378) encompassing nucleotides −751 to −1 was PCRamplified under the same conditions, using the primers e2p(Nhe1)-f(5′-GAG GAG GAG GCT AGC CGA TGT GAC TGT CTC CTC CCA AAT TTG TAG ACC-3′)(SEQ ID NO: 34) and e2p(Hind3)-b (5′-GAG GAG GAG AAG CTT GGT GCT CAC TGCGGC TCC GGC CCC ATG-3′) (SEQ ID NO: 35). PCR products were purified withthe Qiagen PCR prep kit, digested with the restriction endonucleasesNhe1 and Hind3, and cloned into pGL3basic (Promega).

An erbB-2 promoter fragment encompassing nucleotides −1571 to −24 wasexcised from pSVOALΔ5′/erbB-2(N-N) by Hind3 digestion and subcloned intopGL3basic. pSVOALΔ5′/erbB-2(N-N) was a gift from Gordon Gill.

Luciferase Assays

For all transfections, HeLa cells were plated in 24 well dishes and usedat a confluency of 40-60%. Typically, 200 ng reporter plasmid(pGL3-promoter constructs or, as negative control, pGL3basic) and 20 ngeffector plasmid (zinc finger constructs in pcDNA3 or, as negativecontrol, empty pcDNA3) were transfected using the lipofectamine reagent(Gibco BRL). Cell extracts were prepared approximately 48 hours aftertransfection. Luciferase activity was measured with the Promegaluciferase assay reagent, in a MicroLumat LB96P luminometer (EG&GBerthold).

Selection Strategy for the Generation of Six-Finger Proteins with DNABinding Specificity

Based on the modular nature of zinc finger domains, as well as the factthat each zinc finger recognizes 3 bp of DNA sequence, severalstrategies can be employed to generate zinc finger proteins, withpreferably one to three fingers, with desired DNA binding specificity.For instance, in vitro evolution of a six-finger protein binding an 18bp target sequence can follow the strategy outlined in FIG. 1. Thetarget sequence is divided into six 3 bp sub-sites, A-F. In the firststep, a Zif268-based zinc finger phage display library in which thecentral finger 2 is randomized is selected against all 6 subsites in thecontext of the 2 wild type fingers. After successful generation of allthe finger 2 variants required for a given target, cDNAs encodingthree-finger proteins recognizing either half-site 1 (ABC) or half-site2 (DEF) are constructed via PCR overlap extension. Finally, standardcloning procedures are used to construct a gene encoding a six-fingerprotein recognizing the whole 18 bp target site.

As an alternative to the serial connection of F2 domain variants, three-and six-finger proteins can be produced by “helix grafting”. Theframework residues of the zinc finger domains, those residues thatsupport the presentation of the recognition helix, vary betweenproteins. The framework residues play a role in affinity andspecificity. Thus, amino acid positions −2 to 6 of the DNA recognitionhelices are either grafted into a Zif268 (Pavletich et al. (1991)Science 252:809-817) or an Sp1C framework (Desjarlais et al. (1993)Proc. Natl. Acad. Sci. U.S.A. 90:2256-2260).

Choice of Human Integrin β₃ and erbB-2 Target Sequences

Panning experiments carried out previously indicated that zinc fingersbinding to G-containing triplets, with a G or a T in 5′-position, aremore readily obtained than zinc fingers binding other triplets. The zincfinger target sequences were selected such that they contained one ormore G's in each triplet of the 18 bp sequence, and that each tripletstarted with a G or a T (Table 2). To conform with these requirements,erbB-2 target B2 was split into two halves separated by two bases. Alonger linker peptide between the appropriate zinc fingers may also forrecognition of such a split site. Blast sequence similarity searcheswere carried out with each of the target sequences and confirmed thateach 18 bp sequence specifies a unique site in the human genome (maximalsimilarity tolerated: 16/18 bp identity).

Since transcription factor AP-2 is involved in deregulated expression oferbB-2 in a significant fraction of ErbB-2 overexpressing tumor celllines, erbB-2 target site B2 was designed to overlap with the AP-2binding site GCTGCAGGC, with the intention of inhibiting expression ofErbB-2 not only as a result of active transcriptional repression, butalso by competition with an important transcription factor. In contrast,zinc finger proteins binding the other erbB-2 target sites (i.e. erbB-2target sites C and D), affect transcription as a result of theireffector domains.

Integrin β3 target sequences B and C2 were chosen at various distancesfrom the transcription start site, to allow for a comparison of theefficacy of transcriptional regulation. Since the selected zinc fingerproteins are fused to transcriptional effector domains (Sadowski et al.,(1988) Nature 335:563-564; Margolin et al. (1994) Proc. Natl. Acad. Sci.USA 91:4509-4513); Sgouras et al. (1995) EMBO 14:4781-4793; Ayer et al.(1996) Mol. Cell. Biol. 16:5772-5781), binding of a zinc finger proteinper se have an effect on the level of transcription.

A list of chosen target sequences for the selection of zinc fingerproteins is given in Table 2, below. Since zinc finger proteins makebase contacts predominantly with one strand of the DNA double helix,only the relevant strand of the target sequence is listed and designated+/− with respect to the coding strand. The location of the targetsequences and position relative to the major transcription start site(s)is given.

TABLE 2 Chosen Target Sequences For The Selection Of Zinc FingerProteins LOCATION SEQ ID Integrin β3 (B3) target sequences B3B GCC TGAGAG GGA GCG GTG −strand, promoter region, −160 bp 72 B3C2 GGA GGG GACGCG GTG GGT −strand, promoter region, −70 bp 73 ErbB-2 (E2) targetsequences E2B2 GTG TGA GAA(CG)GCT GCA GGC +strand, promoter, −150/−220bp 74 E2B2 GTG TGA GAA(CG)GCT GCA GGC +strand, promoter, −150/−220 bp 74E2C GGG GCC GGA GCC GCA GTG +strand, 5′ UTR, +160/+230 bp 75 E2D GCA GTTGGA GGG GGC GAG +strand, promoter, −30/−100 bp 76

Construction and Panning of a Finger 2 Library

The amino acid residues implicated in contacting DNA in finger 2 of theZif268-C7 (“C7”) (Wu et al. (1995) Proc. Natl. Acad. Sci. U.S.A.92:344-348) have been extensively randomized using the PCR overlapextension mutagenesis strategy. Using two different randomizationstrategies, two sublibraries have been constructed using the pComb3phage display vector (Barbas et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:7978-7982). The sublibraries contain approximately 4×10⁹ independentclones each.

The mutagenesis strategy for randomization of finger 2 of Zif268-C7,showing helix positions −3 to 7, is summarized in Table 3, below. Thetop line shows the wild type sequence of finger 2. The lower two linesshow the two mutagenesis strategies used, where N=G, A, T, C; K=G, T;V=G, A, C; S=G, C. The NNK randomized codon provides all 20 amino acidsin 32 codons. The VNS randomized codon provides 16 amino acids in 24codons, excluding Phe, Trp, Tyr, Cys and all stops. Note that in thestrategy shown in the bottom line, the use of less complex codons allowsfor the mutagenesis of an additional codon.

TABLE 3 Mutagenesis strategy for randomization of finger 2 of Zif268-C7,showing helix positions −3 to 7. −3 −2 −1 1 2 3 4 5 6 7 F S R S D H L TT H F S (NNK) (NNK) (NNK) (NNK) L (NNK) (NNK) H F (VNS) (VNS) (VNS)(VNS) (VNS) L (VNS) (VNS) H

Finger 2 variants recognizing each of the 16 triplets of the GXX set(Segal et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2758-2763; andTable 1), as well as one variant recognizing TGA, have been successfullyselected. In extension of previous observations, comparison of the zincfinger sequences revealed a code for zinc finger recognition of DNA.Thus, a 5′- and 3′-G selected an arginine at helix positions 6 and −1,respectively, while a central G selected an a histidine or lysine atposition 3 of the recognition helix. In contrast, a central A selectedan asparagine, a 3′-A a glutamine, a central T a serine or alanine, a3′-T a threonine or serine, a central C an aspartate or threonine, and a3′-C an aspartate or glutamate at the corresponding helix positions. Anextensive characterization of the specificities and affinities ofselected zinc finger variants has been carried out and indicates thatmany of the zinc finger peptides recognize their targets in a highlyspecific manner (Segal et al. (1999) Proc. Natl. Acad. Sci. U.S.A.96:2758-2763 and Table 1).

Refinement of Finger 2 Specificities by Site-Directed Mutagenesis

Attempts were made to improve binding specificity of some of the zincfinger domains by modifying the recognition helices by usingsite-directed mutagenesis. Data from the phage display selections andstructural information guided the design of the mutants. Although helixpositions 1 and 5 were not expected to play a direct role inrecognition, the best improvements in specificity always involvedmodifications in these positions (Segal et al. (1999) Proc. Natl. Acad.Sci. U.S.A. 96:2758-2763 and Table 1). These residues have been observedto make phosphate backbone contacts, which contribute to affinity in anonsequence-specific manner. Thus, removal of nonspecific contacts canincrease the importance of the specific contacts to the overallstability of the complex, thereby enhancing specificity.

Generation of Three Finger Proteins Binding erbB-2 and Integrin β3Target Sequences

Two different strategies for generating three-finger proteinsrecognizing 9 bp of DNA sequence were used. Each strategy is based onthe modular nature of the zinc finger domain, and takes advantage of afamily of zinc finger domains recognizing triplets of the 5′-GNN-3′ typedefined in Table 1. Two three-finger proteins recognizing half sites(HS) 1 and 2 of the 5′-(GNN)₆-3′ erbB-2 target site e2c were generatedin the first strategy by fusing the pre-defined finger 2 (F2) domainvariants together using a PCR assembly strategy.

To examine the generality of this approach, three additionalthree-finger proteins recognizing sequences of the 5′-(GNN)₃-3′ type,were prepared using the same approach. Purified zinc finger proteinswere prepared as fusions with the maltose binding protein (MBP). ELISAanalysis revealed that serially connected F2 proteins were able to actin concert to specifically recognize the desired 9-bp DNA targetsequences (Beerli et al. (1998) Proc. Natl. Acad. Sci. U.S.A.95:14628-14633). Each of the 5 proteins shown was able to discriminatebetween target and non-target 5′-(GNN)₃-3′ sequence. The affinity ofeach of the proteins for its target was determined by electrophoreticmobility-shift assays. These studies demonstrated that the zinc fingerpeptides have affinities comparable to Zif268 and other naturaltranscription factors with K_(d) values that ranged from 3 to 70 nM(Table 4, below).

As an alternative to the serial connection of F2 domain variants, in thesecond strategy, three-finger proteins specific for the two halfsites ofthe erbB-2 target site e2c (Table 4, below), were produced by “helixgrafting.” The framework residues may play a role in affinity andspecificity. For helix grafting, amino acid positions −2 to 6 of the DNArecognition helices were either grafted into a Zif268 (Pavletich et al.(1991) Science 252:809-817) or an Sp1C framework (Desjarlais et al.(1993) Proc. Natl. Acad. Sci. U.S.A. 90:2256-2260). The Sp1C protein isa designed consensus protein shown to have enhanced stability towardschelating agents. The proteins were expressed from DNA templatesprepared by a rapid PCR-based gene assembly strategy. In each case,ELISA analysis of MBP fusion proteins showed that the DNA bindingspecificities and affinities (Table 4, below) observed with the F2framework constructs were retained. Three finger proteins recognizingHS1 and HS2 of the integrin β3 target sites b3b and b3c2 have also beengenerated, using the Sp1C backbone. Preliminary ELISA data showed thatthese proteins bind their respective targets with good specificity.Further characterization of proteins can be made, such as determinationof their affinities by gel shift analysis. See Table 4, below.

Generation of Six-Finger Proteins for Specific Targeting of the erbB-2and Integrin β3 Promoter Regions.

The recognition of 9 bp of DNA sequence is not sufficient to specify aunique site within a complex genome. In contrast, a six-finger proteinrecognizing 18 bp of contiguous DNA sequence could define a single sitein the human genome, thus fulfilling an important prerequisite for thegeneration of a gene-specific transcriptional switch. Six-fingerproteins binding the erbB-2 target sequence e2c were generatedthree-finger constructs by simple restriction enzyme digestion andcloning with F2, Zif268, and Sp1C framework template DNAs (for sequencesof these proteins, see Beerli et al. (1998) Proc. Natl. Acad. Sci.U.S.A. 95:14628-14633). Six finger proteins binding the integrin β3target sequences b3b and b3c2 were only generated using the Sp1Cbackbone. ELISA analysis of purified MBP fusion proteins showed thateach of the six-finger proteins was able to recognize the specifictarget sequence, with little cross reactivity to non-target 5′-(GNN)₆-3′sites or a tandem repeat of the Zif268 target site.

In Table 4, below, the affinities of three- and six-finger proteins forvarious target sequences as determined by gel shift analysis issummarized. Proteins are named with upper case letters, DNA targetsequences with lower case letters. Abbreviations used are: F2=finger 2framework; Zif=Zif268 framework; Sp1=Sp1C framework; mut=mutant;HS=half-site. With respect to the target site overlap phenomenon, thebase following each target sequence is given in lower case letter (seeBeerli et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633). Theaffinity of the Zif268-DNA interaction was determined to be 10 nM (Segalet al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2758-2763). K_(d) valuesare averages from 2 independent experiments, with standard deviations of50% or less.

TABLE 4 AFFINITIES OF THREE AND SIX FINGER PROTEINS Protein TargetTarget Sequence (5′-3′) K_(d), nM B3(F2) b3 GGA GGG GAC g 4 E2(F2) e2GGG GGC GAG g 3 C5(F2) c5 GGA GGC GGG g 30 E2C-HS1(F2) e2c-hs1 GGG GCCGGA g 45 E2C-HS1(Zif) e2c-hs1 GGG GCC GGA g 70 E2C-HS1(Sp1) e2c-hs1 GGGGCC GGA g 35 E2C-HS2(F2) e2c-hs2 GCC GCA GTG g 70 E2C-HS2(Zif) e2c-hs2GCC GCA GTG g 75 E2C-HS2(Sp1) e2c-hs2 GCC GCA GTG g 25 E2C(F2) e2c-g GGGGCC GGA GCC GCA GTG g 25 E2C(Zif) e2c-g GGG GCC GGA GCC GCA GTG g 1.6E2C(Zif) e2c-a GGG GCC GGA GCC GCA GTG a 2.3 E2C(Zif) e2c-muths1 AGT CTGAAT GCC GCA GTG g 200 E2C(Zif) e2c-muths2 GGG GCC GGA AGT CTG AAT g 200E2C(Sp1) e2c-g GGG GCC GGA GCC GCA GTG g 0.5 E2C(Sp1) e2c-a GGG GCC GGAGCC GCA GTG a 0.75 E2C(Sp1) e2c-muths1 AGT CTG AAT GCC GCA GTG g 65E2C(Sp1) e2c-muths2 GGG GCC GGA AGT CTG AAT g 100

In Table 5, below, the finger 2 variants generated by phage displayselection and refined by site-directed mutagenesis are summarized.Protein designations are in the form pXXX, for clones derived frompanning; pmXXX refers to clones refined by mutagenesis. Helix positions−1, 3, and 6 are shown in bold, altered nucleotides are underlined. Thevalues represent the results of at least two independent experiments.The standard error was ±50% or less.

TABLE 5 SUMMARY OF FINGER 2 VARIANTS GENERATED BY PHAGE DISPLAYSELECTION AND REFINED BY SITE-DIRECTED MUTAGENESIS Finger-2 Finger-2K_(D), _(Prot)/ Protein Helix Subsite K_(D) (nM) K_(D), _(Zif268) pGGGSRSDHLTR GGG 0.4 0.04 pmGGG SRSDKLVR GGG 6 0.6 pmGGG SRSDKLVR GTG >1,400pGGA SQRAHLER GGA 3 0.3 pmGGT STSGHLVR GGT 15 1.5 pmGGT STSGHLVRGGC >2,400 pmGGC SDPGHLVR GGC 40 4.0 pmGAG SRSDNLVR GAG 1 0.1 pmGAGSRSDNLVR GGG 45 4.5 pmGAA SQSSNLVR GAA 0.5 0.05 pGAT STSGNLVR GAT 3 0.3pmGAC SDPGNLVR GAC 3 0.3 pmGAC SDPGNLVR GCC 90 9.0 pGTG SRKDSLVR GTG 30.3 pmGTG SRSDELVR GTG 15 1.5 pmGTG SRSDELVR GAG 30 3.0 pGTA SQSSSLVRGTA 25 2.5 pGTA SQSSSLVR GTG >1,000 pmGTT STSGSLVR GTT 5 0.5 pGTCSDPGALVR GTC 40 4.0 pGTC SDPGALVR GCC >4,400 pmGCG SRSDDLVR GCG 9 0.9pmGCG SRSDDLVR GAG 6 0.6 pGCA SQSGDLRR GCA 2 0.2 pGCA SQSGDLRR GCT 10 1pmGCT STSGELVR GCT 65 6.5 pGCC SDCRDLAR GCC 80 8.0 pTGA SQAGHLAS TGA ndnd C7 SRSDHLTT TGG 0.5 0.05 Zif268 SRSDHLTT TGG 10 1

The affinity of each of the E2C proteins for the e2c DNA target site wasdetermined by gel-shift analysis. A modest K_(d) value of 25 nM wasobserved with the E2C(F2) six-finger protein constructed from the F2framework (Table 5, above; Beerli et al. (1998) Proc. Natl. Acad. Sci.U.S.A. 95:14628-14633), a value that is only 2 to 3 times better thanits constituent three-finger proteins. In previous studies of six-fingerproteins, an approximately 70-fold enhanced affinity of the six-fingerproteins for their DNA ligand compared to their three-fingerconstituents was observed (Liu et al. (1997) Proc. Natl. Acad. Sci.U.S.A. 94:5525-5530). The absence of a substantial increase in theaffinity of the E2C(F2) peptide suggested that serial connection of F2domains is not optimal. It is possible that the periodicity of the F2domains of the six-finger protein does not match that of the DNA overthis extended sequence, and that a significant fraction of the bindingenergy of this protein is spent in unwinding DNA. In contrast to the F2domain protein, the E2C(Zif) and E2C(Sp1) six-finger proteins displayed40- to 70-fold increased affinity as compared to their originalthree-finger protein constituents, with K_(d) values of 1.6 nM and 0.5nM, respectively. Significantly, both three-finger components of theseproteins were involved in binding, since mutation of either half-siteled to a roughly 100-fold decrease in affinity (Table 4, above; Beerliet al (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633). Thepreponderance of known transcription factors bind their specific DNAligands with nanomolar affinity, suggesting that the control of geneexpression is governed by protein/DNA complexes of unexceptional lifetimes. Thus, zinc finger proteins of increased affinity should not berequired and could be disadvantageous, especially if binding tonon-specific DNA is also increased. The affinities of the B3B(Sp1) andB3C2(Sp1) six finger proteins for their respective targets can bedetermined by one skilled in the art using well-known methods as well asthose described herein.

EXAMPLE 2 Construction of Fusion Proteins Containing Zinc Finger Domainsand Transcriptional Repressors and Activators

In order to demonstrate use of zinc finger proteins as gene-specifictranscriptional regulators, the E2C(Sp1), B3B(Sp1), and B3C2(Sp1)six-finger proteins were fused to a number of effector domains (Beerliet al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633).Transcriptional repressors were generated by attaching either of threehuman-derived repressor domains to the zinc finger protein. The firstrepressor protein was prepared using the ERF repressor domain (ERD)(Sgouras et al. (1995) EMBO J. 14:4781-4793), defined by amino acids 473to 530 of the ets2 repressor factor (ERF). This domain mediates theantagonistic effect of ERF on the activity of transcription factors ofthe ets family. A synthetic repressor was constructed by fusion of thisdomain to the C-terminus of the zinc finger protein.

The second repressor protein was prepared using the Krüppel-associatedbox (KRAB) domain (Margolin et al. (1994) Proc. Natl. Acad. Sci. USA91:4509-4513). This repressor domain is commonly found at the N-terminusof zinc finger proteins and presumably exerts its repressive activity onTATA-dependent transcription in a distance- and orientation-independentmanner, by interacting with the RING finger protein KAP-1. The KRABdomain found between amino acids 1 and 97 of the zinc finger proteinKOX1 was used. In this case an N-terminal fusion with the six-fingerprotein was constructed. Finally, to demonstrate the utility of histonedeacetylation for repression, amino acids 1 to 36 of the Mad mSIN3interaction domain (SID) were fused to the N-terminus of the zinc fingerprotein (Ayer et al., (1996) Mol. Cell. Biol. 16:5772-5781). This smalldomain is found at the N-terminus of the transcription factor Mad and isresponsible for mediating its transcriptional repression by interactingwith mSIN3, which in turn interacts the co-repressor N-CoR and with thehistone deacetylase mRPD1.

To examine gene-specific activation, transcriptional activators weregenerated by fusing the zinc finger protein to amino acids 413 to 489 ofthe herpes simplex virus VP16 protein (Sadowski et al. (1988) Nature335:563-564), or to an artificial tetrameric repeat of VP16's minimalactivation domain, DALDDFDLDML (SEQ ID NO: 36) (Seipel et al. (1992)EMBO 13:4961), designated VP64.

Specific Regulation of erbB-2 Promoter Activity

Reporter constructs containing fragments of the erbB-2 promoter coupledto a luciferase reporter gene were generated to test the specificactivities of the erbB-2 specific synthetic transcriptional regulators.The target reporter plasmid contained nucleotides −758 to −1 withrespect to the ATG initiation codon, whereas the control reporterplasmid contained nucleotides −1571 to −24, thus lacking all but onenucleotide of the E2C binding site encompassed in positions −24 to −7.Both promoter fragments displayed similar activities when transfectedtransiently into HeLa cells, in agreement with previous observations. Totest the effect of zinc finger-repressor domain fusion constructs onerbB-2 promoter activity, HeLa cells were transiently co-transfectedwith each of the zinc finger expression vectors and the luciferasereporter constructs (Beerli et al., (1998) Proc. Natl. Acad. Sci. U.S.A.95:14628-14633). Significant repression was observed with eachconstruct. The ERD and SID fusion proteins produced approximately 50%and 80% repression, respectively. The most potent repressor was the KRABfusion protein. This protein caused complete repression of erbB-2promoter activity. The observed residual activity was at the backgroundlevel of the promoter-less pGL3 reporter. In contrast, none of theproteins caused significant repression of the control erbB-2 reporterconstruct lacking the E2C target site, demonstrating that repression isindeed mediated by specific binding of the E2C(Sp1) protein to itstarget site. Expression of a zinc finger protein lacking any effectordomain resulted in weak repression, approximately 30%, indicating thatmost of the repression observed with the SID and KRAB constructs iscaused by their effector domains, rather than by DNA-binding alone. Thisobservation strongly suggests that the mechanism of repression is activeinhibition of transcription initiation rather than of elongation. Onceinitiation of transcription by RNA polymerase II has occurred, the zincfinger protein appears to be readily displaced from the DNA by theaction of the polymerase.

The use of erbB-2 specific zinc finger proteins to mediate activation oftranscription was demonstrated using the same two reporter constructs.The VP16 fusion protein was found to stimulate transcriptionapproximately 5-fold, whereas the VP64 fusion protein produced a 27-foldactivation. This dramatic stimulation of promoter activity caused by asingle VP16-based transcriptional activator is exceptional in view ofthe fact that the zinc finger protein binds in the transcribed region ofthe gene. This again demonstrates that mere binding of a zinc fingerprotein, even with one with sub-nanomolar affinity, in the path of RNApolymerase II need not necessarily negatively affect gene expression.

Based on the efficient and specific regulation of a reporter constructdriven by the erbB-2 promoter, the effect of transiently transfectedzinc finger expression plasmids on activity of the endogenous erbB-2promoter was analyzed. As a read-out of erbB-2 promoter activity, ErbB-2protein levels were analyzed by Western blotting. Significantly,E2C(Sp1)-VP64 lead to an upregulation of ErbB-2 protein levels, whileE2C(Sp1)-SKD lead to its downregulation. This regulation was specific,since no effect was observed on expression of EGFR.

It is important to note that the observations made in these experimentsdrastically underestimate the efficacy of the zinc finger peptides,since the transfection efficiency of HeLa cells is no more than 50%. Toascertain that 100% of the cells express the zinc finger proteins stablecell lines need to be generated. Production of stable cell linesexpressing the zinc finger constructs under control of atetracycline-inducible promoter is known (Gossen et al. (1992) Proc.Natl. Acad. Sci. U.S.A. 89:5547-5551). Inducible expression of zincfinger proteins in stable cell lines allows for detailed analysis of thedegree of specificity of such proteins.

Specific Regulation of Integrin β3 Promoter Activity

To test the activity of transcriptional regulators specific for theintegrin β3 promoter, a reporter plasmid was constructed containing theluciferase open reading frame under control of the integrin β3 promoter.When compared to the two erbB-2 promoter fragments described above, theintegrin β3 promoter fragment had a very low activity. In fact, in someexperiments no activation of luciferase expression over background wasdetected, preventing an analysis of the effects of the KRAB fusionproteins. However, when the VP64 fusion proteins were tested anefficient activation of the integrin β3 promoter was observed.B3B(Sp1)-VP64 and B3C2(Sp1)-VP64 stimulated transcription 12 and22-fold, respectively. Activation of transcription was specific, sinceno effect on the activity of the erbB-2 promoter was detected.

EXAMPLE 3 Fusion Protein Construct Comprising Progesterone ReceptorVariant

Amino acid sequence comparisons of steroid receptor family membersindicates that they generally comprise a number of defined domains,including an N-terminal DNA binding domain and a more C-terminallylocated ligand binding domain. Importantly, these domains are modularand the DNA binding domain of progesterone receptor (PR) has beensuccessfully exchanged for the Gal4 DNA binding domain. The addition ofa VP16 activation or a KRAB repressor domain to the N- or C-terminus ofthis construct yielded proteins that could regulate a Gal4 responsivereporter in a ligand dependent manner. An important feature of theligand binding domain used in these studies is that it is derived from amutant PR with a small C-terminal deletion. This mutant fails to respondto progesterone and is responsive only to progesterone antagonists suchas RU486, making this system suitable for in vivo applications.

The original PR DNA binding domain can be replaced by engineered zincfinger proteins. For example, the three finger protein Zif268(C7) wasfused to the N-terminus of the PR ligand binding domain (PBD) (aa 640 to914), and the VP16 activation domain to its C-terminus. It was foundthat this fusion protein was able to regulate an SV40 promoterluciferase construct with ten upstream Zif268(C7) binding sites in anRU486-dependent manner.

An RU486 dose response curve showed that optimal induction occurs atabout 1 nM to about 10 nM RU486. A time course study was carried outwith 10 nM RU486 and showed that optimal induction of C7-PBD-VP16activity occurs at about 24 hours.

Since naturally occurring steroid receptors bind DNA as dimers, animportant prerequisite for the application of this approach is thepresence of suitable target sequences in the promoter of interest.Fortunately, the spacing and orientation of the two half-sites targetedby steroid receptor dimers is flexible. While a steroid response elementusually includes an inverted repeat, or palindrome, also direct repeatsor even everted repeats of the half-sites in variable spacing aretolerated (Aumais et al. (1996) J. Biol. Chem. 272:12229-12235). Asearch of the erbB-2 and integrin β3 promoters revealed that direct andinverted repeats of 5′-(GNN)₃-3′ sequence motifs occur quite frequently.An example of a sequence motif suitable for targeting by a heterodimericRU486-regulatable zinc finger protein is 5′ GAG GAG GGC TGCTT GAG GAAGTA-3′ (SEQ ID NO: 37), which was found in the erbB-2 promoter andoverlaps with the TATA box (underlined above). In some instances,promoter targeting is possible using a homodimer, for example bytargeting the sequence 5′-GCC GGA GCC ATGGG GCC GGA GCC-3′ (SEQ ID NO:38), which is also found in the erbB-2 promoter and overlaps with thetarget sequence e2c (underlined).

EXAMPLE 4 Recombinant Ligand Activated Transcriptional Regulator FusionProteins Containing Human Estrogen Receptor Ligand Binding Domains

The human estrogen receptor is shown in FIG. 2 as an example of asteroid receptor protein. The numbers below the rectangle indicate theposition of the amino acid residues defining the borders of each domain.A/B is the domain of the amino terminus activation function 1 (AF-1), Cis the DNA binding domain, D is called the hinge region, E is the ligandbinding domain, which also contains the activation function 2 (AF-2) andF is the portion closest the carboxyl terminal, a domain whose functionhas not been fully established. The regions of the protein thatparticipate and stabilize the homodimerized complex are distributed inthe C, D and E domains. Regions throughout the steroid receptor ligandbinding domain (region E in FIG. 2) as well as regions in the native DBDand hinge region (regions C and D respectively) contribute tohomodimerization of the receptor. To demonstrate the importance of theseregions to the function of the C2H2-containing receptors, proteinscontaining three different length LBD fragments were constructed. Thesediffering length LBD constructs are designated A, B, and C (FIG. 3). LBDfragment A represents what is generally referred to as the “minimal” LBDfragment. Some studies have suggested the hinge region plays animportant role in steroid receptor LBD—chimeric proteins; fragment Brepresents the LBD plus hinge. The native C or DNA binding region ofestrogen receptor contains two zinc fingers of the C4-C4 class. The 5′or amino terminus finger contributes to DNA specific contacts; the 3′finger contributes to stabilizing the DNA binding domain dimer complex.To take advantage of this contribution of the 3′ native zinc finger, LBDfragment C, where the 3′ native zinc finger is retained and fuseddirectly to the C2H2 zinc finger array, was included.

In order to optimize the ability of the fusion proteins to regulate geneexpression, it may be necessary to add additional heterologoustransactivating domains to the receptor. To facilitate these studies,fusion proteins were constructed either with the full length LBDextending to estrogen receptor residue 595, or with LBD fragmentstruncated at amino acid (aa) 554 to remove the F region. The full-lengthconstructs are referred to as long (L), the truncated versions as short(S). All constructs contain a heterologous transactivation domain (TA)comprised of a VP16 minimal domain, unless otherwise noted, fused to thecarboxy terminus of the ligand binding domain. VP16 minimal domaintrimer has the amino acid residue sequence 3 x (PADALDDFDLDML) (SEQ IDNO: 47), and is the tetracycline controlled transactivator (tTA) TA2(Baron et al. (1997) Nucleic Acids Research 25:2723-2729).

These constructs are summarized in FIG. 3, which provides a schematicsummary of the cloning strategy and nomenclature related to the C2H2 DNAbinding domain—ER ligand binding domain fusion proteins. As shown in theplasmid construct at the bottom, the final construct contains threecomponents: a C2H2 zinc finger domain (ZFP) at the amino end, a steroidreceptor ligand binding domain (LBD) fragment in the middle, and aheterologous transactivation domain (TA) appended onto the carboxyl end.LBD fragments A, B, or C were defined by the position of the aminoterminus border of the LBD; amino acid number for A (283), B (258) and C(212) correspond to the residue numbers in wild type ER. LBD fragmentswere further defined as long (L) or short (S) depending on their carboxyterminus junction. Long constructs fuse the heterologous TA to the wt ERamino acid residue 595, short constructs fuse TA to an LBD fragmenttruncated at ER amino acid 554. Thus, six fusion proteins in all wereconstructed, ZFP-LBD-TA A, B and C, each in a long and short form. Mapsof specific examples constructed in the expression vector pcDNA3.1 areshown in FIG. 4 (C7LBDAS) (SEQ ID NO: 6), FIG. 5 (C7LBDBS) (SEQ ID NO:8), FIG. 6 (C7LBDCS) (SEQ ID NO: 10), FIG. 7 (C7LBDAL) (SEQ ID NO: 7),FIG. 8 (C7LBDBL) (SEQ ID NO: 1), and FIG. 9 (C7LBDCL) (SEQ ID NO: 9).

As discussed in detail above, zinc fingers of the C2H2 class eachcontribute to about 3 bp of DNA sequence contacts. C2H2 zinc fingerarrays can be “stitched together” to assemble DNA binding domains having6, 9, 12, 15, 18 bp or more of specific sequence to which they bind. Inorder to evaluate the size of the zinc finger array that can be used inthese C2H2 Zn finger (ZFP)—steroid receptor fusion proteins, proteinscontaining 3 finger and 6 finger arrays were constructed. Thecomposition of the various proteins assembled, and their DNA bindingsite specificity is listed in FIG. 16.

The general cloning strategy was as follows. Three fragments (A, B, andC with reference to FIG. 3) of human estrogen receptor ligand bindingdomain (LBD) with or without the F region were built into the pcDNA3.1(Invitrogen) vector backbone through a series of PCR amplification andcloning steps. Initially the LBD fragment A without F region (i.e. shortform; LBDAS) and with F region (i.e. long form; LBDAL) were PCRamplified from a plasmid clone of the human wild type estrogen receptor,pHEGO (Tora et al. EMBO J. 8:1981-1986) with primer pairs NR1/NR2 andNR1/NR3 respectively (Table 1). Convenient restriction sites wereincorporated into primers (Table 1) as needed. The PCR amplified LBDASand LBDAL fragments were first cloned into the Srf I site ofpCR-ScriptAmpSK(+) vector (Stratagene), resulting in constructs pLBDASand pLBDAL. The VP16 minimal domain trimer (TA2; Baron et al. (1997)Nucleic Acids Research 25:2723-2729) was PCR amplified from plasmidpTTA2 (Clontech) with primer pairs NR4 and NR9 and cloned into the SplIand NotI site of pLBDAS and pLBDAL to generate pLBDASTA2 and pLBDALTA2.To generate LBD fragment B without the F region (LBDBS) and LBD fragmentC without the F region (LBDCS), PCR primers NR7 and NR8, which representthe 5′ boundary of the LBD region fragment in chimerics B and Crespectively were designed (Table 6, below). These primers were pairedwith the 3′ end primer NR6, which incorporates a unique BlpI site in ER.PCR fragments from pHEGO with primer pair NR6/NR7 and PCR fragment withNR6/NR8 were then cloned into the SpeI and BlpI site of pLBDC7ASTA2backbone. This resulted in plasmid pLBDBSTA2 and pLBDCSTA2.

TABLE 6 PCR Primers Used For Cloning NAME/(SEQ ID NO:) SEQUENCE NR1 (39)cct act gcc ggc act agt tct gct gga gac atg aga gct gcc aac ctt NR2 (40)cct aaa cgt acg gct agt ggg cgc atg tag gcg gtg ggc gtc NR3 (41) cct aaacgt acg gac tgt ggc agg gaa acc ctc tgc ctc NR4 (42) cca ctt aaa tgt gaaagt cgt acg ccg gcc NR6 (43) tat ggg ggg ctc agc atc caa caa ggc act NR7(44) cct act act agt gac cga aga gga ggg aga atg ttg aaa cac aag cgc NR8(45) cct act act agt agt att caa gga cat aac gac tat atg tgt NR9 (46)tat cat gtg cgg ccg ctt act tag tta ccc cgg cag cat

Having completed cloning of the three LBD fragments fused to the TA2region, the C2H2 DNA binding protein C7 was then excised frompcDNAC7VP16 by BglII and SpeI digestion and ligated into the BamHI andSpeI site of each of the 3 constructions (pLBDASTA2, pLBDBSTA2 andpLBDCSTA2), which resulted in pC7LBDASTA2, pC7LBDBSTA2 and pC7LBDCSTA2.Cassettes of C7LBDASTA2, C7LBDBSTA2 and C7LBDCSTA2 were then removedfrom the pCR-Script vector by EcoRI-NotI digestion and cloned into thesame sites of the expression cassette vector pcDNA3.1(+), resulting inconstructs pcDNAC7ASTA2, pcDNAC7BSTA2 and cDNAC7CSTA2. In order toreconstruct these three ZFP-LBD fusion proteins with an LBD fragmentincluding the estrogen receptor F region fused to TA2, the BlpI to NotIfragment was excised from pLBDALTA2 construct and substituted for theBlpI-NotI fragment in pcDNAC7LBDASTA2, pcDNAC7LBDBSTA2 andpcDNAC7LBDCSTA2 to generate pcDNAC7LBDALTA2, pcDNAC7LBDBLTA2 andpcDNAC7LBDCLTA2.

Cloning for Replacement of DNA Binding Domain C7 with E2C

An intermediate construct pcDNAE2CVP16 was first constructed byreplacing the SfiI fragment containing C7 in pcDNAC7VP16 with theE2C(hs1) fragment isolated from pMal/E2C(hs1) after SfiI digestion.Next, pcDNAE2CVP16 was digested with SpeI and a 1 kb fragment wasisolated. This SpeI fragment was ligated to the large SpeI fragment ofpcDNAC7LBDASTA2, which created pcDNA-E2CLBDASTA2. Similar steps wereperformed to construct pcDNAE2CLBDBSTA2.

Analysis of Recombinant Construct Protein Binding to DNA

In order to demonstrate that the fusion proteins bind to DNA in asequence specific manner, and to evaluate the stoichiometry ofprotein:DNA binding, standard electrophoretic mobility shift or gelretardation assays were performed.

First, fusion proteins were produced by in vitro transcription andtranslation using the TNT Coupled Reticulocyte Lysate System (Promega,Cat #L4610) according to the manufacturer's instructions. Briefly, eachexpression reaction was set up in a total volume of 50 μl whichcontained 25 μl of TNT rabbit reticulocyte lysate, 2 μl of TNT ReactionBuffer, 2 μl of RNasin ribonuclease inhibitor (20 U/μl), 1 μl each ofamino acid mixture minus leucine, amino acid mixture minus methionineand TNT T7 RNA polymerase, 2 μl of expression plasmid (1 μg/μl) andwater. The reaction mixture was incubated at 30° C. for 90 minutes.

Binding of the expressed protein to duplex oligonucleotides wasperformed as follows, using the gel shift assay systems (Promega, Cat#E3050): 5 μl of in vitro translation product was co-incubated with 4 μlof 5× gel shift binding buffer and 7 μl of water at room temperature for20 min, then 2 μl of E2 (10 nM final concentration) and 2 μl of³²P-labeled probe were added to the mixture. The probe had been labeledusing standard protocol as described in the kit. After incubated at roomtemperature for about 20 minutes, the mixture was loaded onto a 6% DNAretardation gel and run in 0.5×TBE buffer at 150-200 volts for about30-60 minutes. The gel was then dried and exposed to X-ray film.

A DNA oligonucleotide containing two inverted binding sites for the C2H2domain known as C7, each half site separated by 3 bp, was used for theinitial assessment of DNA binding. This palindromic configuration mimicsthe composition of the native estrogen receptor response element (ERE),except that the natural 6 bp half site of ERE is replaced by the 9 bphalf site specified by C7. Binding of the C7-LBD fusion proteins A, B,and C, all in the short form, were tested and compared to the controlproteins C7VP16 and 2C7VP16 (see, Liu, et al. (1997) Proc. Natl. Acad.Sci. U.S.A. 94:5525-5530, which describes the control proteins). Foreach protein, binding was tested in the absence or presence of 100 foldexcess of unlabeled oligonucleotide (1.75 μM) as a competitor.Competition of the gel shift product by the unlabeled oligonucleotideindicates the band is a specific protein:DNA interaction. The resultsdemonstrated that C7VP16 can bind once or twice to the oligonucleotide,creating two specific gel shift bands. 2C7VP16 binds only once to theoligonucleotide containing two inverted C7 sites. Notably, C7LBDA andC7LBDB bind strongly to yield one major species, which runs higher thanany of the control bands. Although true molecular mass cannot bedetermined from this type of mobility assay, the relative size of thecomplexes suggest the protein bound for C7LBD is larger than for C7VP or2C7VP. The size of the band and presence of only one major speciesindicate that the fusion protein ZFP-LBD is binding to theoligonucleotide as a dimer. No significant gel shift product wasdetected for C7LBD chimeric C, suggesting that the addition of theadditional native zinc finger from the estrogen receptor may havereduced the affinity of the fusion protein for its C2H2-specific DNAbinding site. Finally, the reduction of binding for each of the gelshift products by the addition of the unlabeled oligonucleotideindicates that these fusion proteins are binding to DNA in a sequencespecific manner.

To further demonstrate that the chimera ZFP-LBD binds to DNA as a dimer,the binding of C7LBD A, B, and C to oligonucleotides containing one ortwo C7 binding sites was tested. Three fusion proteins (C7LBDAS, C7LBDBSand C7LBDCS) were tested against three different target oligonucleotidesequences, which contained one C7 half site or two C7 half sites eitherin palindromic or direct repeat orientation.

Oligo 1: gat cca aag tcg cgt ggg cgc agc gcc cac gcg atc aaa ga (SEQ IDNO: 48) Oligo 2: gat cca aag tcc agg cga gcg cgt ggg cgg cag atc aaa ga(SEQ ID NO: 49) Oligo 3: gat cca aag tcg cgt ggg cgc agg cgc gag cgt gggcgg atc aaa ga (SEQ ID NO: 50)

Gel shift assay conditions were the same as the standard protocoldescribed above. The results showed that C7LBDAS and C7LBDBS were ableto bind to both oligonucleotides containing two C7 half sites, but notto the oligo containing only one half site. C7LBDCS bound weakly or notat all to all three targets.

Fusion proteins C7LBDA and C7LBDB bound to the probe containing apalindrome (two inverted half sites) as a single form and in equalamount to the C7VP control, while C7LBDC showed no detectable binding.In contrast, the fusion proteins C7LBDA and C7LBDB did not bind to theoligonucleotide containing only one C7 site, while C7VP bound only once,as expected. C7LBDA and C7LBDB bound equally to the oligo 1 and oligo 3,which contain two sites as inverted repeats with 3 intervening spaces ordirect repeats with 9 intervening spaces, respectively. These dataindicate that the ZFP-LBD fusion proteins dimerize and bindpreferentially to DNA containing two C7 half sites, but that the exactorientation and spacing of the half sites is not critical. Thisflexibility in DNA binding site orientation may reflect the lack of adimerization function in the C2H2 domains, but it is noteworthy thatwild type estrogen receptor has also been shown to bind a variety ofresponse elements differing from the consensus ERE, including invertedand direct repeats.

To further confirm the homodimer binding stoichiometry of the ZFP-LBDfusion proteins and to demonstrate their DNA sequence specificity, thefollowing experiment was conducted. A second ZFP-LBD fusion protein wasconstructed using the C2H2 zinc finger domain E2C(HS1), which binds to arecognition sequence 5′-GGG GCC GGA g 3′ that differs in six out of ninebase pairs from the C7 binding site. (Note that the lower case g denotesa 10^(th) base that makes a minor contribution to the protein:DNAcontact affinity.) Maps of specific examples constructed in theexpression vector pcDNA3.1 are shown in FIG. 11 (E2CLBDAS) (SEQ ID NO:11) and FIG. 12 (E2CLBDBS) (SEQ ID NO: 12).

Oligonucleotides were prepared containing an inverted repeat of two C7sites, two E2C sites, or a mixed heterodimeric site of one C7 and oneE2C half site. Two fusion proteins having different DNA binding domains(C7 or E2C) were tested for their DNA binding specificity against threeoligonucleotides containing palindromic binding sites specific for C7,E2C or the combination of the two.

C7 oligo: gat cca aag tcg cgt ggg cgc agc gcc cac gcg atc aaa ga (SEQ IDNO: 51) C7/E2C oligo: gat cca aag tcg cgt ggg cgc act ccg gcc ccg atcaaa ga (SEQ ID NO: 52) E2C oligo: gat cca aag tcg ggg ccg gag act ccggcc ccg atc aaa ga (SEQ ID NO: 53)Gel shift assays were performed according to the standard protocoldescribed above.

The results showed that C7LBD fusion protein only binds strongly to theoligonucleotide containing two C7 sites, but not to either the 2×E2Cprobe or the C7/E2C probe. Likewise, the E2C-LBD chimeric protein onlybinds strongly to the 2 x E2C probe. Finally, neither ZFP-LBD constructbinds to the oligonucleotide with the heterodimeric site. When the twoproteins were mixed in equal amount, a C7LBD and E2CLBD heterodimer wasformed. The heterodimer binds to the heterodimeric probe. These resultsconfirm that the ZFP-LBD fusion proteins are binding DNA preferentiallyas dimers. Furthermore, these data demonstrate good DNA bindingspecificity between fusion proteins with different C2H2 binding sitepreferences.

EXAMPLE 5 Ligand-Dependent Regulation of Transgene Expression by ZFP-LBDFusion Proteins

In order to evaluate the ability of the fusion proteins C7LBD A, B, andC to regulate transgene expression, a standard co-transfection reporterassay was performed. A reporter construct, henceforth known as6X2C7pGL3Luc, containing six copies of a directly repeated C7 bindingsite (6X2C7) inserted upstream of an SV40 promoter fragment and reportergene encoding firefly luciferase (pGL3Pro; Promega) was transfectedalong with the designated fusion protein and assayed as described below.

Cultured cells (HeLa, Cos, Hep3B or other) were seeded at 5×10⁴cells/well in a 24 well plate prior to the day of transfection in DMEMPhenol-free media, supplemented with L-glutamine and 5% (v/v)charcoal-dextran stripped Fetal Bovine serum (sFBS). Cells weretransfected using the Qiagen Superfect Transfection method. For eachwell 1 μg of total DNA, containing 0.5 μg luciferase reporter plasmid(6X2C7pGL3proluc), 0.1 μg of chimeric activator DNA (e.g., C7LBDA,C7LBDB, or C7LBDC) unless otherwise indicated, and 0.4 μg of an inertcarrier plasmid DNA (p3Kpn), was mixed with 60 μL of DMEMphenol-free/serum free media, and 5 μL of Superfect reagent. In general,about 10 ng to about 0.5 μg of chimeric activator DNA was used for eachwell.

The mixture was vortexed for 10 seconds and incubated at roomtemperature for 10 minutes, followed by the addition of 350 μL of DMEMphenol-free 5% sFBS media. Cells were washed once with Dulbecco'sphosphate buffered saline (DPBS) and the transfection mixture placed onthe cells. Cells were washed once with DPBS following a 2.5 hourincubation at 37 degrees Celsius, and re-fed with DMEM Phenol-free 5%sFBS media.

At approximately 24 hours post-transfection, cells were treated with aninducing agent, 17β-Estradiol or 4 OH-Tamoxifen as indicated, each at100 nM final concentration in DMEM Phenol-free 5% sFBS. Cells wereharvested 24 hours later by washing once with DPBS and adding 200 μL 1×reporter lysis buffer (Promega). Plates were frozen at −80° C. andthawed at room temperature for 1.5 hours on an orbital shaker at 100RPM. After allowing for cellular debris to settle, lysate was diluted1:10 with 1× reporter lysis buffer, and 10 μL transferred to 96 wellopaque plates. Plates were analyzed with a Tropix TR717 MicroplateLuminometer using firefly luciferase substrate (Promega).

The ability of C7LBD short form chimeric proteins A, B, and C toregulate reporter gene expression in an estrogen-dependent manner wasstudied in Cos and HeLa cells. The constitutive activators C7VP16 and2C7VP16 were used as positive controls. The results show that the threeZFP-LBD fusion proteins gave a similar profile in Cos and HeLa cells.All three ZFP-LBD fusion proteins had an estrogen dependent effect onthe luciferase reporter gene. The characteristic pattern is that A hasgreater total activity than B and B has greater total activity than C.Likewise, the basal or ligand-independent effect of these proteins onthe reporter gene follows a similar pattern: A>B>C. The estrogendependent effect on gene expression ranged from two-fold to nine-fold inthese experiments.

The regulation of luciferase reporter gene by the C7LBD long and shortform fusion proteins was compared in Cos cells. The results indicatethat the long form fusion proteins, which contain the estrogen receptorF region, have a higher basal and ligand-independent effect on thereporter gene than the short form. As a result, the long fusion proteinsgive lower fold induction. This result may be due to an enhanced, butligand-independent, transactivation activity in the F region that workssynergistically with the heterologous VP minimal domain trimer.Alternatively, this result could be due to the difference in spacing, asa result of the intervening F region, between the VP activation domainand the estrogen receptor ligand binding domain of the recombinantproteins.

In order to evaluate the role of the composition of the heterologoustransactivation domain on the activity of the C7LBD fusion proteins, theVP minimal domain trimer was replaced with either the carboxy terminalactivation domain from human STAT-6 (amino acids 660-847) or the fulllength VP16 activation domain of approximately 77 amino acids (residues413-490) (FIG. 13). In constructs with full length VP16, thetransactivation domain was added either native, or in conjunction withan SV40 nuclear localization peptide sequence at the amino terminus ofthe VP16. C7LBD fusion proteins A or B containing differenttransactivation domains (TA2, STAT6C, VP16 and NLSVP16) were constructedand evaluated for their effects on gene activation and ligand induction.The construct, shown schematically and abbreviated above, includes thefollowing:

1. C7ASTA2, C7BSTA2: C7LBD A or B short form with the VP16 minimaldomain trimer.2. C7BS-STAT: C7LBDB short form with the STAT6 carboxy activationdomain.3. C7BS VP16: C7LBDB short form with full length VP16 activation domain.4. C7AS nlsVP16: C7LBDA short with full length VP16 preceded by a nls.

Assays were performed with HeLa cells transfected with 0.5 ug of6X2C7pGL3Luc reporter and 0.1 μg regulator, Luc activity was determinedas previously described. When the human STAT6 transactivation domain wasused to replace the TA2 VP minimal domain trimer, the same low basalactivity and 9 fold ligand dependent induction of transgene, two-foldless than with the TA2 domain, was obtained.

The incorporation of NLS upstream of the full length VP16 (FIG. 24,C7ASnlsVP16) greatly increased the folding induction compared to TA2 orVP16 without the NLS, but the total activity was significantlydecreased. When the full length VP16 domain was used, it gave about 2fold higher total activity, but high basal activity resulting in weakerligand dependent induction (3-fold).

EXAMPLE 6 Ligand-Independent Activity of C7-PBD-VP16 Constructs Dependson the Structure of the Reporter Constructs

In initial tests, the C7-PBD-VP16 construct showed the high basal (i.e.ligand-independent) activity. Thus, C7-PBD-VP16 was compared to theoriginal, Gal4-based construct GL914VPc′, which reportedly had a verylow basal activity. When the GL914VPc′ protein was tested on a6×Gal4-SV40 promoter-luciferase reporter, it displayed even higher basalactivity than C7-PBD-VP16. Variation of effector/reporter ratios had noeffect on the basal activities in both systems. It was discovered,however, that the ratios for optimal induction were different forGL914VPc′ and C7-PBD-VP16, namely 1/30 and 1/10, respectively.

Other possible sources of ligand-independent activity were examined.Commercially available fetal calf serum (FCS) batches are known tocontain estrogen or estrogen-like activities. Since it was possible thatthe presence of progesterone-agonistic activities in the serum was thecause for the high basal activities, the FCS was “stripped” of steroidsusing dextran-coated charcoal. However, side-by-side comparison ofstripped and non-stripped serum showed no detectable difference in thebasal activity of the switch constructs. Lipid-based transfectionreagents such as Lipofectamine™ can also have significant agonisticactivity on steroid receptors. Thus, the non-lipid transfection reagentSuperfect™ from Qiagen was used as an alternative, and compared toLipofectamine™. No reduction of the basal activities was observed. Forall the assays described above, HeLa cells were used. However, the useof HepG2 cells, which were used in the original study with GL914VPc′,brought no improvement.

The reporter p17x4TATA-luc, used in the original studies on Gal4,contains four Gal4 dimer binding sites upstream of a TATA box. GL914VPc′had a very low basal activity on this reporter, and was inducible byRU486. An equivalent reporter, pGL3TATA/10xC7, was therefore constructedto test C7-PBD-VP16. While the basal activity using a reporter constructhaving TATA reporter was still higher than in the Gal4 system, basalactivity was clearly lower than using the SV40 promoter-containingpGL3prom/10xC7. Two additional reporters with minimal CMV promoters,pGL3minCMV/6×Gal4 and pGL3minCMV/10xC7, were also constructed. The basalactivity of the corresponding switch proteins was as high on thesereporters as on the SV40 promoter containing reporters.

These results indicate that GL914VPc′ and C7-PBD-VP16 wereconstitutively located in the nucleus and able to bind to their targetsites, either as monomers or as dimers. However, unless bound to ligandthe fusion proteins are only able to activate transcription in thecontext of more than a TATA box, i.e. a SV40 promoter or a minimal CMVpromoter. If there is only a TATA box, ligand binding presumablyassociated with a conformational change is required for efficientactivation of transcription.

It was found that ligand-independent basal activity is also cell typespecific. C7-PBD-VP16 had an even lower basal activity on the TATAreporter in NIH/3T3 cells than it had in HeLa cells.

Since C7-PBD-VP16 appears to be constitutively translocated to thenucleus, the SV40 nuclear localization signal (NLS) between PBD and VP16domains was removed in the hope of making nuclear translocation moreligand dependent. The resulting construct, C7-PBD-VP16noNLS, was thentested on the pGL3prom/10xC7 reporter. However, transcriptionalactivation was no more RU486-dependent than in the case of C7-PBD-VP16as shown by an unchanged basal activity. The construct C7-PBDΔNLS-VP16noNLS was made in which the small remaining part of a naturalSV40-like NLS at the N-terminus of the PBD (aa 640-644) is also removed.

EXAMPLE 7 Optimizing Spacing and Orientation of the DNA Binding DomainHalf-Sites

Naturally occurring steroid hormone receptors typically bind to aninverted repeat, or palindromic SRE. However, it has been shown inseveral cases that there is some flexibility binding. Direct repeats andeverted repeats can also serve as response elements. To determine theoptimal spacing and orientation of the two half-sites for binding of asteroid receptor-based switch construct a total of eighteen C7 dimerTATA-luciferase reporter constructs were prepared. Six C7 dimers each indirect, inverted and everted repeat orientation, with spacers of 0 to 5intervening bases. A test of the RU486-responsive C7-PBD-VP64 protein oneach of these reporter constructs revealed that indeed there was quitesome flexibility, since RU486 inducible activation was observed witheach of the reporters (Tables 7-9, below; values listed are means of twodeterminations and the standard deviation). There were clear differencesin the degree of responsiveness of each of the reporters.

A direct repeat of two C7 sites without any spacing displayed the mostfavorable properties. This is particularly important, indicating theability to target (GNN)₆ sites using homodimeric and heterodimericrecombinant ligand-responsive transcription factors.

Further tests on the RU486-responsive VP64-C7-PR protein and thetamoxifen-responsive VP64-C7-ER protein, on each of these reporterconstructs also revealed some flexibility, since ligand-inducibleactivation was observed with each of the proteins on each of thereporters. However, the most favorable properties were observed with theVP64-C7-ER protein on the direct and everted repeats with a spacing of 3bp. Direct repeat with a spacing of 5 bp was also more or lessreasonable, permitting targeting of the erbB-2 promoter with a 3 fingerconstruct (see below).

Further studies have shown that binding of a C7/Cf2-PBD-VP64 heterodimerto a C7-Cf2 TATA reporter, with one binding site each for C7 and Cf2without spacing, provides about a two-fold ligand-dependent change intranscription.

TABLE 7 Direct Repeats C7-PBD-VP64 Mean STD DEV C7c7 4081 511 C7c7 +RU486 20018 2090 C7ac7 3383 396 C7ac7 + RU486 8205 2064 C72ac7 3417 348C72ac7 + RU486 8169 634 C73ac7 3269 1550 C73ac7 + RU486 5138 2319 C74ac73966 298 C74ac7 + RU486 6945 1377 C75ac7 2597 416 C75ac7 + RU486 5460207

TABLE 8 Inverted Repeats C7-PBD-VP64 Mean STD DEV C77c 2921 1368 C77c +RU486 10811 1596 C7a7c 4342 153 C7a7c + RU486 9534 2943 C72a7c 6964 573C72ac7 + RU486 19186 3284 C73a7c 7132 5208 C73a7c + RU486 12844 171C74a7c 3502 416 C74a7c + RU486 8855 2379 C75a7c 4704 105 C75a7c + RU48612444 2117

TABLE 9 Everted Repeats C7-PBD-VP64 Mean STD Dev 7cc7 8750 1839 7cc7 +RU486 17377 1335 7cac7 6029 613 7cac7 + RU486 13599 2014 7c2ac7 78801720 7c2ac7 + RU486 20825 8197 7c3ac7 9670 1187 7c3ac7 + RU486 21491 2747c4ac7 6974 441 7c4ac7 + RU486 8896 2455 7c5ac7 6892 388 7c5ac7 + RU48613124 3490

EXAMPLE 8 C7-PBD-Repressor Domain Fusion Constructs

To evaluate the use of PBD fusion proteins as regulatabletranscriptional repressors, C7-PBD was fused to a number of repressordomains (Table 10, below). When tested in luciferase reporter assays,many repressor constructs had no significant activity. C7-PBD-KK(containing a dimer of two KRAB-A boxes) reproducibly led to a 25-50%repression, which was largely RU486-dependent. A much strongerrepression which, however, was largely RU486-independent was observedwith a C7-PBD-SKD construct.

EXAMPLE 9 Regulation of erbB-2 Promoter Activity with ThreeFinger-PBD-VP64 Homo-/Hetero-Dimers

The C7-PBD-VP16 switch protein was able to regulate 10xC7 reporterconstructs, which contain 10 direct repeats of C7 sites with a spacingof 5 bp (see above), indicating that a switch dimer can bind to directrepeats with this specific spacing. To evaluate the potential use ofhomo- and hetero-dimeric three finger-PBD fusion proteins for theligand-dependent regulation of erbB-2 promoter activity, the promoterregion was screened for the presence of (GNN)₃N₅(GNN)₃ motifs. Fourdimer target sites (E2E, E2F, E2G, and E2H) were identified. E2Eoverlaps with the 18 bp E2C target sequence and could serve as a bindingsite for a homodimer. The other three sites have the potential to serveas heterodimer binding sites. The seven required three finger proteinswere generated by F2 stitchery and analyzed for binding by ELISA (Table11, below). erbB-2-specific switch constructs were then generated byfusion of each three finger protein to PBD-VP64, and tested for theirability to regulate erbB-2 promoter activity. The values are mean andstandard deviation of duplicate measurements. Only the heterodimericE2F-PBD-VP64 switch led to a detectable regulation of the erbB-2promoter. This regulation was not RU486-dependent, consistent with thehigh basal activities of C7-PBD-VP16 and C7-PBD-VP64 proteins.

TABLE 10 Progesterone Receptor Based Ligand-Responsive TranscriptionFactors Transcription Effector DNA Binding Domain Ligand Binding DomainDomain C7 hPR (aa 640-914) VP16 C7 hPR (aa 640-914) VP64 C7 hPR (aa640-914) KRABa C7 hPR (aa 640-914) Mad C7 hPR (aa 640-914) Mad-Mad C7hPR (aa 640-914) KRABa-Mad C7 hPR (aa 640-914) Mad-KRABa C7 hPR (aa640-914) Deactylase C7 hPR (aa 640-914) SKD 2C7 hPR (aa 640-914) VP162C7 hPR (aa 640-914) VP64 E2E 3F hPR (aa 640-914) VP64 E2F 3F hPR (aa640-914) VP64 E2G 3F hPR (aa 640-914) VP64 E2H 3F hPR (aa 640-914) VP64E2C(SP1) 6F hPR (aa 640-914) VP16 E2C(SP1) 6F hPR (aa 640-914) VP64E2C(SP1) 6F hPR (aa 640-914) KRABa E2C(SP1) 6F hPR (aa 640-914) MadE2C(SP1) 6F hPR (aa 640-914) KRABa-KRABa E2C(SP1) 6F hPR (aa 640-914)Mad-Mad E2C(SP1) 6F hPR (aa 640-914) KRABa-Mad E2C(SP1) 6F hPR (aa640-914) Mad-KRABa

TABLE 11 Mean STD DEV Mean STD DEV Target Basal Basal RU486 RU486 TargetSequence Binding Activity Activity Activity Activity Control pcDNA 3.117209 1878 E2C-HS1 ggg-gcc-gga good E2C-HS2 gcc-gca-gtg good E2Egcc-gga-ggc none 18259 140 15893 2083 E2F-HS1 gag-gag-ggc good 6140125291 54986 19240 E2F-HS2 gag-gaa-gta ? E2G-HS1 ggg-gcc-ggg weak 259825444 12394 139 E2G-HS2 ggc-gca-gta weak E2H-HS1 ggc-gcg-ggg weak 15374844 15374 537 E2H-HS2 ggt-gct-gcg none

EXAMPLE 10 Estrogen and Progesterone Receptor Fusion Proteins withN-Terminal Effector Domains

Recombinant ligand-responsive polypeptides were constructed using anestrogen receptor (ER) ligand binding domain (EBD). A Myc-ER fusionconstruct was obtained from Eliane Muller and used as a source of theEBD coding region. Rather than containing the human wild type amino acidsequence, Myc-ER contains a point mutation (aa 282-599, G525R) mouse EBDwhich has been shown to no longer bind estrogen, but bind the estrogenantagonist 4-OH tamoxifen, and paradoxically becomes activated by it.This has advantages for in vivo applications and for tissue cultureexperiments, not only because serum contains estrogen but also becausephenol red present in all tissue culture media acts as an estrogenagonist.

The VP16-C7-ER, VP 16-NLS-C7-ER, and VP16-C7-NLS-ER fusion constructswere prepared as described above. In parallel, an analogous set ofprogesterone receptor (PR) variants was also prepared (VP16-C7-PR,VP16-NLS-C7-PR, and VP16-C7-NLS-PR. The PBD in these constructsencompasses aa 640-914 and therefore lacks the partial natural NLS (aa640-644).

Each of these constructs was tested in a luciferase assay and comparedto C7-PBD-VP16, using pGL3prom/10xC7 as a reporter. Not only did allthese PR constructs have a higher activity in the presence of RU486 thanC7-PBD-VP16, but the completely NLS-free VP16-C7-PR also had asignificantly lower basal activity. This resulted in a dramaticallyimproved ligand-dependent induction, 26-fold vs. 6-fold in thisparticular experiment. Tamoxifen-induced activity of the ER constructswas roughly four times higher than RU486-induced activity of the PRvariants. Ligand-dependent induction was better; 43 fold for VP16-C7-ER.

The VP16 domain in VP16-C7-PR and VP16-C7-ER has been replaced by thefollowing effector domains: the activator VP64, and the repressors KK(KRAB-A box dimer), MM (dimer of the Mad sin3 interaction domain) andSKD. The VP64 variants are useful, for example, in studies to determinethe optimal spacing and orientation of the two half-sites, using theabove-mentioned C7 dimer-TATA luciferase reporters

EXAMPLE 11 Targeting Natural Promoters Using 3 Finger Proteins Fused toNuclear Hormone LBDs

The following target sequences for 3 Finger switch homo- andhetero-dimers have been identified in the human erbB-2 (E2) and integrinβ3 (B3) promoters:

E2E (SEQ ID NO: 54) GCC`GGA`GCC ATGGG GCC GGA GCC direct repeat, 5 bpspacing homodimer B3D (SEQ ID NO: 55) CGC TCC CTC TCA GGC GCAGGG everted repeat, 3 bp spacing, heterodimer B3E (SEQ ID NO: 56) GGCGCC CAC TGT GGG GCG GGC everted repeat, 3 bp spacing, heterodimer.

EXAMPLE 12 Targeting Natural Promoters Using Six Finger Proteins Fusedto Nuclear Hormone Ligand Binding Domains The “6 Finger Heterodimer”

Regulation of a 6 finger protein binding to a single 18 bp site usingany of the formats described have been unsuccessful. Similarly, aC7-PBD-VP64 protein did not activate a TATA reporter containing only asingle C7 site. As an alternative, heterodimer constructs were preparedin which only one of the dimerization partners contains a DNA bindingdomain, while the other contains an effector domain.

The formats were as follows:

(1) E2C-PR//PR-VP64 (2) E2C-ER//ER-VP64

All four fusion constructs were fully sequenced and tested in aluciferase assay for their ability to regulate the erbB-2 promoter in aligand-dependent manner. It was found that the PR 6 Finger heterodimerwas inactive; a similar observation was made with an C7-RxR//EcR-VP16heterodimer. In contrast, the E2C-ER//ER-VP64 heterodimer had someactivity, and the addition of Tamoxifen lead to a roughly three-foldupregulation of promoter activity. Variations in the ratio of the twoheterodimerization partners led to an increased inducibility, up tototal of 5.3-fold.

The coding region for RXR (mammalian) and EcR (Drosophila) were PCRamplified from pVgRXR (Invitrogen) using the primers listed below andAmpliTaq DNA Polymerase (Hoffmann-LaRoche). Forward and backward primerswere chosen to allow construction of the constructs. The cyclingconditions were 2′/94° C.; 25×(300″/94° C.-30″/60° C.-2′/72° C.);10′/72° C. The PCR product was purified with the Quiagen PCR prep kit,cut with the indicated restriction endonucleases and ligated into amodified eukaryotic expression vector pcDNA3 (Invitrogen; see, also,Beerli et al (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633) toyield the constructs in FIG. 14.

Primers: (FseI)-RXR: (SEQ ID NO: 57)GAGGAGGAGGGCCGGCCGGGAAGCCGTGCAGGAGGAGCGGC RXR-(AscI): (SEQ ID NO: 58)GAGGAGGAGGGCGCGCCCAGTCATTTGGTGCGGCGCCTCCAGC RXR-(PacI): (SEQ ID NO: 59)GAGGAGGAGTTAATTAAAGTCATTTGGTGCGGCGCCTCCAGC (FseI)-EcR: (SEQ ID NO: 60)GAGGAGGAGGGCCGGCCGGGGTGGCGGCCAAGACTTTGTTAAGAAGG (SfiI)-EcR: (SEQ ID NO:61) GAGGAGGAGGGCCCAGGCGGCCGGTGGCGGCCAAGACTTTGTTAAGAAGG EcR-(AscI): (SEQID NO: 62) GAGGAGGAGGGCGCGCCCGGCATGAACGTCCCAGATCTCCTCGAG

Exchange of Zinc Finger and Effector Domains

After digestion with the restriction endonuclease SfiI the C7 3-fingerprotein was replaced with the 6-finger proteins E2C, B3B, B3C2 and 2C7by standard cloning procedures. After digestion with the restrictionendonucleases AscI and PacI the activation domain VP16 was replaced withthe activation domain VP64 and the repression domains KK and SKD.

Luciferase Assays

For all transfections, HeLa cells were plated in 24-well dishes and usedat a confluency of 40-60%. Typically, 175 ng reporter plasmid(pGL3-promoter constructs or, as negative control, pGL3basic) and 25 ngeffector plasmid (zinc finger constructs in pcDNA3 or, as negativecontrol, empty pcDNA3.1) were transfected using the Lipofectaminereagent (Gibco BRL). Cell extracts were prepared approximately 48 hoursafter transfection. Luciferase activity was measured with the Promegaluciferase assay reagent in a MicroLumat LB96P luminometer (EG&GBerthold).

Bombyx mori EcR

A plasmid (LNCVBE) containing the coding region for Bombyx mori EcR wasobtained from F. Gage. Bombyx mori EcR is PCR amplified from thisplasmid using the primers listed below and AmpliTaq DNA Polymerase(Hoffmann-LaRoche). Forward and backward primers were chosen to allowconstruction of the constructs corresponding to FIG. 14 but replacingDrosophila EcR by Bombyx mori EcR.

(FseI)-BE: (SEQ ID NO: 63)GAGGAGGAGGGCCGGCCGGAGGCCTGAATGTGTCATACAGGAGCCC (SfiI)-BE: (SEQ ID NO:64) GAGGAGGAGGGCCCAGGCGGCCAGGCCTGAATGTGTCATACAGGAGCCC BE-(AscI): (SEQ IDNO: 65) GAGGAGGAGGGCGCGCCCCTCCGCCACGTCCCAGATCTCCTCGAG

C7-R-VP16//C7-E-VP16

This heterodimer was examined on two reporters, one containing 10 C7sites and one containing 6 2C7 sites, and in two cell lines, HeLa andNIH. In all cases the C7-R-VP16 construct alone showed a high activationof transcription (840-fold) that did not depend on the presence ofPonasterone A. However the C7-E-VP16 construct showed a very littleactivation of transcription on its own. C7-R-VP16//C7-E-VP16 togethershowed the same behavior as C7-R-VP16 alone.

C7-R//E-VP16

In this heterodimer, the activation domain on RXR is dropped toeliminate the basal activation observed above. EcR has no DNA-bindingdomain to render activation dependent on the presence of DNA-bound RXR.This heterodimer was tested with the 3-finger protein C7 on the 10C7reporter and with the 6-finger protein E2C on the E2P reporter thatcontains a single E2P binding site. In both cases no significantactivation could be observed.

C7-R//C7-E-VP16

To combine the low basal activity of C7-R//E-VP16 with the highactivation seen with C7-R-VP16//C7-E-VP16, the activation domain on RXRwas dropped but the zinc finger protein on EcR was retained. In thisset-up, on a 6X2C7 reporter, a 5-fold activation with very low basalactivity was observed. Similar constructs using the more powerful VP64activation domain have also been made.

E2C-ER//ER-VP64

This heterodimeric construct showed 5.3 fold tamoxifen-dependentactivation at ratios of 6.7/60 and 2.2/60 of the erbB-2 promoter.

EXAMPLE 13 Regulation of Endogenous ErbB-2 Gene Expression UsingAdenovirus-Mediated Delivery of E2C-KRAB

Adenovirus vectors can be produced at very high titers, which makes themuseful for gene therapy applications. To demonstrate the use of theE2C-KRAB repressor protein in animal models, E2C-KRAB (and, as acontrol, 2C7-KRAB) encoding adenoviruses were generated. The method foradenovirus production is described in detail, for example, in He et al.(1998) Proc. Natl. Acad. Sci. U.S.A. 95:2509-2514.

Briefly, the zinc finger coding regions were excised from thepMX/E2C-KRAB and pMX/2C7-KRAB bicistronic retrovirus plasmids byBamH1H-Not1 digest. The resulting fragments were then subcloned into theBgl2-Not1 sites of pAdTrack-CMV. After linearization with Pme1, pAdTrackplasmids were co-electroporated with circular pAdEasy-1 into BJ5183cells. This bacterial strain is not recA and therefore allows homologousrecombination between the 2 plasmids. Electroporated cells were thenplated onto Kan plates. Only plasmids that have recombined togetherprovide Kanamycin resistance, because this marker is only present onpAdTrack. After screening to distinguish recombinants from background(due to incomplete linearization of pAdTrack plasmids), the linearadenovirus vector genomes were released from the recombinantpAdEasy/E2C-KRAB and pAdEasy/2C7-KRAB plasmids by Pac1 digest.

The linearized vectors were then transfected into 293 cells. This cellline makes the Adeno E1A and E1B proteins, which have been deleted fromthe vector and are required for replication.

EXAMPLE 14 Modifications to the Estrogen Receptor Ligand Binding DomainImprove Ligand Dependent Induction and Ligand Selectivity

Single amino acid mutations in the estrogen receptor ligand bindingdomain can have a significant effect on the basal and ligand dependentlevel of gene activation. For example, a glycine to valine substitutionat estrogen receptor residue 400, has been described as a destabilizingor temperature sensitive mutation (White (1997) Adv. Pharmacol.40:339-367; Aumais et al. (1996) J. Biol. Chem. 272:12229-12235). Theeffect of this mutation on the properties of the fusion proteins wastested. The general methods for constructing fusion proteins withaltered amino acids is described below.

Mutagenesis of the fusion proteins C7LBDa and C7LBDb was performed usingoligonucleotide mediated site directed mutagenesis (Stratagene;Quikchange Site-Directed Mutagenesis Kit) to either substitute argininefor glycine at amino acid 521 (G521R-human estrogen receptornomenclature) or a valine residue for glycine at amino acid 400 (G400V).The sequences of the oligonucleotides used for G521R mutagenesis wereGTACAGATGCTCCATGCGTTTGTTACTCATGTGCC (SEQ ID NO: 66) for the noncodingstrand and GGCACATGAGTAACAAACGCATGGAGCATCTGTAC (SEQ ID NO: 67) for thecoding strand, where the nucleotide in bold represents the change fromthe wild type sequence.

The sequences of the oligonucleotides used for G400V mutagenesis wereCCATGGAGCACCCAGTGAAGCTACTGTTTGC (SEQ ID NO: 68) for the coding strand,and GCAAACAGTAGCTTCACTGGGTGCTCCATGG (SEQ ID NO: 69) for the noncodingstrand, where the nucleotide in bold represents the change in sequencefrom wild type.

Templates were added at 10 ng to 50 ng per reaction with 125 ng of eachprimer in 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.8), 2 mMMgSO₄, 0.1% Triton X-100, 0.1 mg/ml BSA, dNTP mix, and 2.5 U PfuTurbo™DNA polymerase. The reactions were carried out on a Perkin Elmer GeneAmpPCR system 9600 thermal-cycle using an initial temperature of 94 degreesCelsius for 30 seconds to denature the template, followed by 12 cyclesat 95 degrees Celsius for 30 seconds, 55 degrees Celsius for 1 minute,and 68 degrees Celsius for 4 minutes, with a single round of extensionat 72 degrees Celsius for 2.5 minutes. PCR samples were treated with 10U DpnI for 1 hr at 37 degrees Celsius to digest the non-mutagenizedparent template.

DH5α supercompetent Epicurean Coli® XL-1 cells were transformed bycombining 1 μL of the DpnI treated PCR samples with 50 μL of the cellsin chilled Falcon 2059 tubes, incubated on ice for 30 minutes, heatshocked at 42 degrees Celsius for 45 seconds and chilled on ice for 2minutes. A 500 μL aliquot of SOC media pre-warmed to 42 degrees Celsiuswas added to the transformation reaction and incubated for 1 hour at 37degrees Celsius with shaking. The transformed cells were plated onto LBplates containing 100 μg/ml ampicillin and incubated for at least 16hours.

Mutation efficiency was determined by altering a nonsense codon in aβ-galactosidase expression plasmid to glutamine and determiningexpression of β-galactosidase, as evidenced by IPTG/X-Gal plates.Approximately three clones for each mutation were selected forrestriction enzyme digestion to check for template integrity, followedby dideoxynucleotide sequencing of the entire coding frame to confirmthe desired mutation.

C7LBD (short) chimeric regulators A, B, and C with and without the G400Vmutation in the estrogen receptor LBD were compared for their ability toinduce expression of the 6X2C7pGL3Luc. As observed previously, the totalactivity of the three fusion proteins has the relationship A>B>C; thisrelationship was maintained with and without the G400V mutation. Thepattern of basal expression was dramatically altered by the G400Vmutation. The basal or ligand independent effect of the three C7LBDregulators with the G400V mutation is reduced to nearly the level of thereporter plasmid alone. As a result, the fold ligand dependent inductiondramatically increases, for example from 10 fold to 420 fold for C7LBDA.

It has previously been observed with fusion proteins containing anestrogen receptor ligand binding domain, that activity could be inducedby use of not only the natural agonist estrogen (E2) but also syntheticanti-estrogens such as 4-OH tamoxifen (Littlewood et al. (1995) Nucl.Acids Res. 23:1686-1690; Danielian et al. (1993) Mol. Endocrinol.7:234-240). The ability of the C7LBD fusion to be induced by4-OH-tamoxifen was demonstrated.

The results of the study showed the ligand-dependent regulation of aluciferase reporter gene construct in HeLa cells using three recombinantmolecular constructs, C7LBDAS, C7LBDBS, C7LBDCS with and without a G400Vmutation in response to estrogen (E2) and 4 hydroxytamoxifen (OHT). Inparticular, the results showed that fusion proteins C7LBD B and C areinduced equally well by 100 nM tamoxifen or estrogen. For C7LBDA,tamoxifen appears to be approximately two-fold more active than estrogenitself.

Another mutation of interest in estrogen receptor LBD is a glycine toarginine substitution at amino acid 521 of human estrogen receptor. Thismutation has also been described in the mouse estrogen receptor homologat the equivalent site of residue 525. This mutation ablatesresponsiveness of the mutated LBD to estrogen, but still allows thebinding of the anti-estrogen tamoxifen (Littlewood et al. (1995) Nucl.Acids Res. 23: 1686-1690; Danielian et al. (1993) Mol. Endocrinol.7:234-240). The effect of the G521R mutation on the activity of theC7LBD regulators was tested. C7LBDB was compared to C7LBDB (G400V) andC7LBDB (G521R).

The results of the study showed the ligand-dependent regulation of aluciferase reporter gene construct in HeLa cells using three recombinantmolecular constructs: C7LBDBS, C7LBDBS with a G521R mutation and C7LBDBSwith a G400V mutation in response to estrogen (E2) and 4hydroxytamoxifen (OHT). Similar to the effect observed with the G400Vmutation, G521R significantly reduces the basal activity of the fusionprotein regulator. But most importantly, now the C7LBDB(G521R) regulatoris fully activated by 100 nM 4-OH-tamoxifen, but completely inactive inresponse to estrogen. Note that the G400V mutant is still fullyactivated by estrogen and tamoxifen.

To further investigate the effect of the G521R mutation, a series ofdifferent estrogenic compounds were evaluated for their ability toinduce the C7LBD regulators. The activity of 100 nM for four compounds:estrogen (E2) and diethyl-stilbesterol (DES) are estrogenic agonists,4-OH-tamoxifen and raloxifen (Ral) are non-steroidal anti-estrogens, orso-called SERMS (selective estrogen receptor modulators) were compared.

The study tested ligand-dependent regulation of a luciferase reportergene construct in Hep3BL liver cells using recombinant molecularconstructs C7LBDBS with a G521R mutation and C7LBDBS with a G400Vmutation in response to estrogen (E2) diethylstilbesterol (DES),4-hydroxytamoxifen (4-OHT) and raloxifen (Ralox). The results showedthat the G521R mutation selectively eliminates response to the agonists,but the non-steroidal synthetic ligands tamoxifen and raloxifen arestill fully active.

EXAMPLE 15 Effect of the Minimal Promoter Composition on Regulation ofTransgenes by ZFP-LBD Fusion Proteins

The composition of the minimal promoter used in reporter assays candramatically effect the level of gene expression. Likewise, the activityof natural steroid receptors varies on different gene targets dependingon the composition of their promoters. Reporter constructs containing6X2C7 binding sites upstream of a minimal TATA box promoter fragmentderived from the c-fos gene, referred to here as TATA were constructedto show the effect on the level of regulation. C7LBD A and B fusionswithout or with the G400V or G521R mutations were compared. As observedpreviously on the pGL3 SV40 promoter, the G400V and G521R mutationssignificantly decrease the basal activity of the chimeras compared tothose without these mutations. Further, the G521R mutant is selectivelyactivated by tamoxifen. On this weaker minimal promoter, estrogen isonly a weak inducer, while 4-OH-tamoxifen is significantly better. Thiseffect is even more pronounced on C7LBD A compared to B; on chimeraC7LBDA (G400V), tamoxifen is at least 10 fold more active than estrogen.

An experiment was done to directly compare the relative activity of theC7LBD chimeras on reporter constructs containing the stronger pGL3 SV40promoter or the weaker c-fos TATA box promoter.

The results of the study show that the ligand-dependent regulation of aluciferase reporter gene construct expressed from a minimal TATApromoter in Hep3BL liver cells using recombinant molecular constructsC7LBDAS and C7LBDBS with a G400V mutation and C7LBDBS with a G521Rmutation in response to estrogen (E2) and 4-hydroxytamoxifen (OHT).Three important observations can be made: 1) the absolute level ofinduced activity is about 10 fold higher on the SV40 than the TATApromoter 2) the basal activity of the fusions is also about 10 foldhigher on the SV40 than on the TATA promoter, 3) while both promotersshow strong fold induction by tamoxifen (492X on SV40 and 132X on TATA),estrogen is only a strong inducer of the SV40 but not the TATA promoter(177X vs 14X). These results indication that a gene regulation systemusing these fusion proteins can be “tuned” by choice of an appropriateminimal promoter.

Target Selectivity of Different C2H2 DNA Binding Domains

Reporter constructs with 3 copies of direct repeats of the C7 bindingsite (GCG TGG GCG) or E2C binding site (GGG GCC GGA g) inserted upstreamof the promoter region in pGL3Luc were used to evaluate targetspecificity two different ZFP-LBDBs fusion protein regulators. ZFP-LBDBshort fusions were constructed containing either the C7 DNA bindingdomain or the E2C DNA binding domain and tested on the two differentreporter constructs. The study was designed to show the effect of threedirect repeats of either C7 or E2C binding sites inserted upstream ofthe promoter of a luciferase reporter gene construct in HeLa cells onestrogen-dependent gene expression using recombinant molecularconstructs C7LBDBS and E2CLBDBS. Estrogen-dependent induction onlyoccurs when the chimera's DNA binding domain (DBD) matches the bindingsites in the reporter. The E2CLBD chimera shows no increase ofluciferase activity on the 3×2C7 Luc reporter and visa versa for C7LBDon the 3xE2C reporter.

It was previously determined from DNA binding studies that the fusionprotein regulators have an absolute dependence on the presence of twohalf sites within a “response element” in order to bind DNA. In order todetermine the optimal orientation and spacing of the binding sites forgene activation, a series of different reporter constructs wereassembled. In order to determine the optimal target DNA spacing andorientation of the C2H2 binding sites for transgene induction, C7LBDBSwas transfected into HeLa cells and assayed for basal and tamoxifeninduced activity on a series of reporter constructs.

A series of different reporter constructs assembled in order todetermine the optimal target DNA spacing and orientation of the C2H2binding sites for transgene induction, C7LBDBS was transfected into HeLacells and assayed for basal and tamoxifen induced activity on a seriesof reporter constructs diagrammed above. Reporter constructs wereconstructed by cloning double stranded oligonucleotides containing thevarious binding sites into the multiple cloning site of the pGL3Lucreporter. “Response elements” composed of direct, inverted(palindromic), and everted repeats of two C7 binding sites werecompared; each response element was separated by two (2) bp except inthe control 6X2C7, where spacing was 5 bp. Several arrays of directlyrepeated single C7 sites were tested with various spacing. The data showthat direct repeats and everted repeats are preferred over palindromicbinding sites. Further, 6 C7 sites, each separated by 2 bp is comparableto the control element of 6X2C7, even though it contains only half thenumber of individual C7 binding sites.

EXAMPLE 16 Construction and Evaluation of ZFP-LBD Fusion ProteinRegulators Containing Arrays of Six C2H2 Zinc Fingers

Studies were performed to determine if DNA binding domains comprised ofzinc finger arrays binding up to 18 bp of DNA could be substituted forthe normal estrogen receptor DBD. The previous constructs, containingthree finger arrays that bind nine bp are a fairly conservativereplacement of the wild type estrogen receptor ligand binding domainthat binds six bp for each receptor monomer. The possibility exists thatif large DNA binding domains are fused to an LBD fragment, that thesedomains may prevent dimerization via the LBD dimerization domain due tosteric interference. However, since the six finger arrays alreadyprovide high DNA specificity and affinity, dimerization may beunnecessary for the DNA binding and activity of these fusions proteins.Fusion protein regulators were prepared by fusing the 2C7 six fingerarray to the three LBD fragments A, B, and C described above. FIG. 15provides a schematic and description of the cloning step required toassemble 2C7LBDshort A, B, and C.

Protein binding to DNA was analyzed by gel shift assay. Theelectrophoretic studies used 2C7 recombinant molecular constructs usingnative PAGE and SDS PAGE analysis of binding to a DNA probe containingsix 2C7 binding sites. In this experiment, the 2C7VP 16 protein was usedas a control and the P32-labeled DNA probe was the 6X2C7 fragmentexcised from the 6X2C7pGL3Luc. Sufficient 2C7VP protein was added toyield three distinct gel shifted products. When a similar level ofprotein for the 2C7LBD A, B, and C were applied, only a single weak bandwas observed. By comparison to the one and two copies bound bands forthe 2C7VP16 control, the 2C7LBD band position suggests it is binding asa monomer. Furthermore, the weak level of binding compared to the2C7VP16 control suggests the DNA binding affinity of the 2C7 domain issignificantly reduced in the context of the LBD fusion protein. Resultsof in vitro expressed proteins by SDS-PAGE, indicated equal amounts ofproteins expressed and the expected relative increase in size for theLBD A, B, and C forms.

The ability of the 2C7LBD A, B, and C fusion protein chimeric regulatorsto activate expression of the 6X2C7Luc reporter gene were evaluatedessentially as described previously for the C7LBD studies. The resultsof the study show the ligand-dependent regulation of a 2C7 SV40luciferase reporter gene construct in Cos cells using three recombinantmolecular constructs, 2C7LBDAS (SEQ ID NO: 1), 2C7LBDBS (SEQ ID NO: 2),2C7LBDCS (SEQ ID NO: 3), and a positive control, 2C7-Vp16. The resultsare similar to the data evaluating C7LBD in Cos cells. The 2C7LBDregulators give about two fold estrogen dependent induction over basal,with 2C7LBDA>B>C for both the total activation activity and theincreased basal activity relative to reporter plasmid alone. Maps of theadditional constructs are depicted in FIG. 16-FIG. 22.

EXAMPLE 17 Construction and Evaluation of Additional Reporter TransgeneConstructs

An inducible promoter was constructed based on binding sites for the 3Finger protein N1. The promoter contains 5 direct repeats of N1 sitesspaced by 3 bp; the spacing between the 5 repeats is 6 bp. (FIG. 23A)

Luciferase assay. HeLa cells were cotransfected with plasmids encodingthe indicated fusion proteins and the N1 reporter construct. At 24 hlater, the cells were treated with 10 nM RU486 (FIG. 23B) or 100 nMTamoxifen (FIG. 23C), respectively. At 48 h post transfection, cellextracts were assayed for luciferase activity.

Another inducible promoter based on binding sites for the 3 Fingerprotein B3. The promoter contains 5 direct repeats of B3 sites spaced by3 bp; the spacing between the 5 repeats is 6 bp (FIG. 24A).

Luciferase assay. HeLa cells were cotransfected with plasmids encodingthe indicated fusion proteins and the B3 reporter construct. At 24 hlater, the cells were treated with 10 nM RU486 (FIG. 24B), or 100 nMTamoxifen (FIG. 24C), respectively. At 48 h post transfection, cellextracts were assayed for luciferase activity.

EXAMPLE 18 Heterodimer Formation in Presence of Ligand

FIG. 25 shows the results of a luciferase assay showing RU486-inducedformation of functional VP64-C7-PR/VP64-CF2-PR heterodimers. HeLa cellswere cotransfected with the corresponding effector plasmids and TATAreporter plasmids (C7/CF2-dr0, C7 site 5′ to a CF2 site, direct“repeat”, no spacing; C7/C7-dr0, 2 C7 sites, direct repeat, no spacing).At 24 h later, the cells were treated with 10 nM RU486. At 48 h posttransfection, cell extracts were assayed for luciferase activity.

EXAMPLE 19 Construction and Evaluation of the Cys₂-His₂ Zinc FingerDBD-ER LBD Regulators in Adenoviral Vectors

In order to efficiently deliver the two components of the regulatorysystem to mammalian cells, either ex vivo or in vivo, a series ofadenoviral vectors were constructed. These vectors contained either theZFP-LBD fusion protein regulator linked to the immediate early CMVpromoter or the regulatable transgene, linked to the 6X2C7 array of C7binding sites and the minimal promoter from SV40 or c-fos TATA asdescribed previously. The fusion protein regulator vector andregulatable transgene vector are then be mixed at various ratios anddelivered to cells or animals by standard methods.

Construction of an adenovirus vector is routine and generally, theprocedure involves three main steps: first a shuttle plasmid containingthe viral left ITR, viral packaging signal, a promoter element, atransgene of interest linked to the promoter element and followed by apoly adenylation sequence, and some additional DNA sequences, viral ornon-viral, required for recombination is prepared. Second, this left endshuttle plasmid, along with the remainder of the viral genome (i.e. theright end of the vector) are transfected into a host cell and joinedthrough DNA recombination to form a complete vector genome. Thisrecombination step may result from sequence homology between the twovector halves or may be aided by the use of site specific recombinasessuch as Cre and their corresponding LoxP recombination sequences.Finally, the newly formed virus is amplified up and purified in a seriesof steps. The details of the construction of these vectors are brieflydescribed below.

Left End Shuttle Plasmid Construction for ZFP-LBD Fusion ProteinRegulators

Shuttle plasmids containing the left viral ITR, CMV immediate earlypromoter and ZFP-LBD regulator were prepared in the plasmid pAvCVlx(FIG. 26). Note that this vector contains a loxP recombination site justdownstream of the poly adenylation sequence. DNA encoding the intactreading frame for the chimeric regulators C7LBD As(G521R), C7LBDBs(G521R), and C7LBD Bs(G400V) were excised from the appropriate pCDNAconstructions, (see FIGS. 4 and 5 for LBD As and LBD Bs constructsrespectively) by digestion with restriction enzymes EcoRI and Not I. TheZFP-LBD DNA fragments were modified with Klenow to fill in therestriction site overhangs and blunt end ligated into the EcoRV at bp1393 site of pAvCvlx to generate pAvCv-C7LBD As(G521R), pAvCv-C7LBDBs(G521R), and pAvCv-C7LBD Bs(G400V).

Construction of Left End Shuttle Plasmids Containing RegulatableTransgene Cassettes

Two regulatable transgene cassettes were prepared. One contained the6X2C7 binding sites and SV40 minimal promoter fragment linked to theLuciferase transgene as in pGL3 6X2C7-Luc (described in example 5). Thesecond vector contained the 6X2C7 binding sites and c-fos TATA minimalpromoter linked to a cDNA encoding murine endostatin fused to an aminoterminal secretion signal. The complete sequence of this fusion proteinis listed in SEQ ID NOs. 70 and 71.

These vectors were constructed in two steps. First, a fragmentcontaining the CMV promoter and tri-partite leader sequence (TPL) ofpAvCvlx (FIG. 26) was excised by digestion with MluI and BglII, whichcut at bp 473 and 1375 respectively. The restriction site overhangs werefilled in with Klenow. Blunt ended DNA fragments containing the6X2C7-SV40 or 6X2C7-TATA enhancer/promoter regions of the previouslydescribed reporter plasmids were ligated into this backbone to createpAV-6X2C7SV40 and pAV-6X2C7TATA shuttle plasmids. Next, DNA fragmentscontaining the Luciferase or murine endostatin transgenes were ligatedinto the EcoRV site of the appropriate shuttle plasmids to createpAv6X2C7SV40-Luc (lox) or pAv6X2C7TATA-mEndo (lox).

Construction of a Right End Vector Plasmid

To complete the vector construction, a plasmid containing the remainderof the viral vector genome is required. This plasmid, referred to aspSQ3, which is shown in FIG. 27, contains a pBR322-derived backbone,ampicillin resistance gene and the adenovirus serotype 5 genome,beginning at Ad5 bp 3329, through the right ITR, with deletions in theE2a and E3 region as described previously (Gorziglia et al. (1996) J.Virol. 70:4173-4178). In addition, this plasmid has two importantfeatures, a loxP site inserted at the Bam HI site (bp 3156.9) justupstream of the Ad5 sequences, and a Cla I site at the end of the viral5′ ITR. This Cla I site is used to linearize the plasmid and expose theright ITR during vector construction.

Vector Assembly and Propagation

Three adenoviral vectors encoding fusion protein regulators,Av3CV-C7LBDAS(G521R), Av3CV-C7LBDBS(G521R), and Av3CV-C7LBDBS(G400V) andtwo vectors containing regulatable transgenes, Av3SV-LUC andAv3TATA-Endo were constructed. Each vector was generated by a standardprocedure. Briefly, for each vector construct, three plasmids, pSQ3 (predigested with ClaI), the appropriate left end shuttle plasmid (e.g.pAvCv-C7LBD As(G521R), or pAv6X2C7SV40-Luc (lox), pre-digested with NotIand Afl II, and an expression plasmid for the Cre recombinase, pCMV-CRE,were cotransfected at a weight ratio of 3:1:1 into dexamethasone inducedAE1-2a cells (Gorziglia et al.) using Promega's Profection Kit. About 1week after transfection, cells were harvested and lysed by 4 cycles offreeze/thaw. The resulting cell lysate was passed onto freshdexamethasone induced AE1-2a cells and the culture maintained about aweek until cytopathic effect (CPE) was observed. This process wasrepeated several cycles until sufficient material was obtained to purifythe vector by CsCl equilibrium density centrifugation. Once purified,vectors are quantitated by lysing in buffer containing 10 mM Tris, 1 mMEDTA, 0.1% SDS for 15 minutes at 56° C., cooling and reading theabsorbance at 260 nm wavelength (OD260). The OD260 reading is convertedto a virus particle concentration using 1 OD260 unit=1.1×10¹²particles/ml.

Results

In Vitro Regulation with Adenovirus Vectors

The ability to regulate expression of a transgene delivered by anadenovirus vector was demonstrated by the following experiment. HeLacells were infected with a mixture of two adenovirus vectors, onecontaining a fusion protein regulator either (Av3-C7LBD-A(G521R) orAv3-C7LBD-B(G52R), the other containing the 6X2C7SV40-luc cassette. Todetermine the optimal ratio of target vector to effector vector, twodifferent doses of the transgene or target vector (50 or 250 viralparticles per cell) at three different ratios of effector vector (50,250, 750 particles per cell for each target dose) were tested. Twentyfour hours after vector transduction, the cells were treated whereappropriate with 100 nM 4-OH-tamoxifen. Following an additional 24 hrsincubation, the cells were lysed and assayed for luciferase activity.For the Av3CV-C7LBD A(G521R) vector, the data indicate relatively lowlevels of luc expression in the absence of 4-OHT, a strong 4-OHTdependent induction and a dose dependent increase in luc activity asmore fusion protein regulator vector is used. At the highest doses (750particles per cell) of chimeric regulator vector tested,tamoxifen-specific induction of 460 to 560 fold over basal was achievedat target vector doses of 250 and 50 particles per cell, respectively.

The same experiment carried out using the LBD B version of the chimericregulator; Av3CV-C7LBD B(G521R). For this vector, the fold induction andabsolute luciferase activity were about two fold lower than obtainedwith the As-based regulator. These results are consistent with all theprevious transient transfection experiments performed with plasmids.Notably, a first generation of Av3-chimeric regulator vectorsconstructed with the RSV promoter driving the expression of the C7LBDgene did not yield good transgene upregulation of the Av3SV40-Lucvector. Apparently, the expression level from the weaker RSV promoterwas not adequate to produce the necessary levels of fusion protein.

In Vivo Regulation with Adenovirus Vectors

To demonstrate the effectiveness of the C7LBD regulators to controltransgene expression in vivo, a study was designed to evaluate threeimportant variables: 1) the effectiveness of regulators containingeither the G400V or G521R mutations, 2) the ratio of target and effectorvector, and 3) the dose of 4-OHT. The importance of the G400V and G521Rmutations are as follows. While the G521R mutation is selectivelyresponsive to 4-OHT and is not affected by endogenous estrogen, itrequires about a 10-fold higher drug concentration than the G400Vmutation to achieve maximum activity. While the G400V is active at alower dose of 4-OHT, it is also subject to induction by estrogen andcould show higher basal activity in vivo.

Details of the animal study are as follows. On study day 1, C57Bl/6 malemice were given a total adenovirus vector dose of 2×10¹¹ particles viatail vein injection. On day two blood samples were collected, thenanimals were injected i.p. with 200 μl of sunflower seed oil containing5% DMSO and either no, 50 μg, or 500 μg of tamoxifen (Sigma #T56448).Blood samples were collected daily for three days following drugadministration, and on study days 8 and 10. At the completion of thestudy, murine endostatin levels were determined by ELISA (Accucyte Kit,Cytimmune Sciences, Maryland).

The study groups included the following:

Negative Control—2×10¹¹ particles Av3Null, Ad vector with no transgene

Positive Control—2×10¹¹ particles Av3RSV-mEndo, constitutively expressesendostatin from the RSV promoter.

1:1 As521—Received 1×10¹¹ particles of Av3TATA-mEndo and 1×10¹¹particles of Av3Cv-C7LBDAs(G521R); no treatment (basal) or +50 μgtamoxifen.

1:1 Bs400—Received 1×10¹¹ particles of Av3TATA-mEndo and 1×10¹¹particles of Av3Cv-C7LBDBs(G400V); no treatment (basal) or +50 μgtamoxifen.

In addition, groups 5 and 6 were similar to groups 3 and 4, but animalsreceived 0.5×10¹¹ of the Av3TATA-mEndo vector and 1.5×10¹¹ of the C7LBDregulator vector, for a 1:3 ratio of target to effector. Groups 3-6 eachcontained no drug, 50 μg, and 500 μg tamoxifen treatment sub-groups.

The results showed a dramatic induction of murine endostatin followingthe day 2 administration of 50 μg of tamoxifen. The highest level ofinduction was observed on day 3, the day immediately following drugadministration. Compared to the basal level observed on day 3 in the notamoxifen groups, the C7LBDAs(G521R) and C7LBDBs(G400V) regulators gavecomparable fold induction, approximately 17 fold, and comparableabsolute levels of expression, around 1500 ng/ml. In this study, theendogenous murine endostatin levels in an untreated mouse cohort was20±7 ng/ml. The drug-induced endostatin expression rapidly declines byday 5, three days after drug administration, which is presumably due tothe clearance of the tamoxifen and biological half life of theendostatin protein. In contrast, expression in the Av3RSV-mEndotreatment group persists at 200 ng/ml through day 15. In the 1:3 targetto effector ratio groups, tamoxifen-induced expression reached 600-900ng/ml, approximately ½ the level in the 1:1 ratio cohorts. This resultindicates that in vivo, the transgene-containing vector, not the fusionprotein-encoding vector, is limiting for absolute protein expression.Furthermore, endostatin expression in the animals treated with 500 μgtamoxifen was comparable to the animals treated with only 50 μg,indicating that the lower dose of tamoxifen is sufficient to fullyactivate the As(G521R) and Bs(G400V) regulators. Finally, the comparablelow basal level of endostatin observed in the As(G521R) and Bs(G400V)groups suggests that the endogenous level of estrogen in the C57Bl/6mice is not sufficient to induce the estrogen-responsive Bs(G400V)regulator. An elevation in basal endostatin levels observed at days 3-5appeared to be a non-specific effect resulting from adenovirus vectoradministration, since the Av3Null vector has an effect similar to theAv3TATA-mEndo containing groups.

Conclusions

The in vitro and in vivo results shown in this Example, demonstrate thatthe ZFP-LBD fusion proteins can be efficiently delivered via anadenovirus vector and can be expressed in sufficient amounts to providehigh levels of drug-dependent control of a transgene in animals.Furthermore, the data show that the basal level of expression from the6x2C7-minimal promoter constructs tested in an adenovirus vector giverelatively low levels of expression, even when the fusion protein isexpressed in the same cell. Thus, the system is highly drug dependentand allows for substantial regulation of the vector-delivered transgene.Taken together, these data evidence the effectiveness of this system forgene therapy applications.

EXAMPLE 20 Construction and Evaluation of the Cys₂-His₂ Zinc FingerDBD-ERLBD Regulators in Lentiviral Vectors

In order to demonstrate controlled gene expression in an integratedvector system, the regulatory system described in Example 19 with theadenoviral vectors were used to develop a series of lentiviral vectors.These vectors contained either the ZFP-LBD fusion protein linked to theimmediate early CMV promoter or a regulatable transgene (either eGFP orluciferase) linked to the 6X2C7 array of C7 binding sites and either theminimal promoter from SV40 or C-fos TATA. The fusion protein-encodingvector and the regulatable transgene vector can then be used to generatelentiviral vector supernatant. The supernatant can be used to stablytransduced human cells either singly or in parallel. Stable cell linescontaining the integrated vectors can then be induced with theappropriate activating drug (e.g., 4-OH-tamoxifen) and gene expressionis measured as fold induction in the presence and absence of drug.

Construction of Lentiviral Vectors Encoding the ZFP-LBD Fusion Proteinor the Regulatable Transgene.

The generation of lentiviral vectors and vector supernatant involves 3main steps: first a gene or region of interest is inserted into shuttlevector backbone plasmid containing all of the viral cis-elements fortranscription, packaging, reverse transcription, and integration.Second, the lentiviral vector shuttle plasmid is co-transfected intohuman 293 cells along with plasmids providing the packaging functions(gag, pol, and env). Typically the transfections include 10 μg of vectorplasmid, 10 μg of packaging plasmid and 1 μg envelope plasmid (VesicularStomatitis virus G envelope) using a Profection Calcium Phosphatetransfection kit. Third, the culture supernatant containing thelentiviral vector is harvested (between 24 and 48 hours posttransfection) and used to transduce naïve human target cells.

Construction of HIV-1 Based Vectors

An HIV-1-based vector system containing an internal CMV promoter wasconstructed from an infectious HIV-1_(IIIB) provirus cDNA (pHIV-IIIB)The infectious proviral cDNA was generated by PCR from DNA isolated fromH-9 cells chronically infected with HIV-1_(IIIB). The gag/pol and envsequences of pHIVIIIB were removed by digestion and excision of aPstI-KpnI fragment. Replacing the gag/pol and env sequences was aPstI/Kpn polylinker containing unique multiple cloning sites to form theintermediate vector p2XLTR. The Rev response element (RRE) fragment fromHIVIIIB, required for proper vector RNA processing, was inserteddownstream of the truncated gag sequences of p2XTR to form the constructpHIVec. An AseI-XbaI CMV-eGFP reporter fragment derived from pEGFP-N1(Clontech, Palo Alto, Calif.) was cloned into the NdeI-Xba site ofpHIVec to generate pHIVCMVGFP. pHIVCMV-X was generated by removal of theeGFP fragment by KpnI digestion and religation.

Construction of pHIVCMV-C7LBD/A(G521R)

The AS521R(C7LBD/A(G521R) coding fragment derived from C7LBDAS bydigestion with NotI, T4 DNA polymerase fill-in, and EcoRI site wascloned into pHIVCMV-X cloned downstream of the CMV promoter into aEcoRI/SmaI restriction site. As a control for induction, an HIV vectorcontaining a constitutive transactivator and DBD chimera was generated,pHIVCMV-C7VP16. A HindIII-NotI restriction fragment from pcDNA3-C7VP16containing the C7VP16 coding fragment was inserted downstream of the CMVpromoter at the Sma site of pHIVecCMV-X.

Construction of pHIV6X2C7Sv and pHIV6X2C7TATA Luciferase Vectors

A BamHI-XbaI restriction fragment containing the 6X2C7TATA luciferasefragment was isolated from pTATA6X2C7Luc and cloned downstream of theRRE at the SpeI-XbaI restriction sites. A MluI-BstBI restrictionfragment containing the 6X2C7Sv luciferase fragment was isolated frompGL3-6X2C7SvLuc and cloned downstream of the RRE at the Spe-XbaIrestriction sites.

Evaluation of the ZFP-LBD Fusion Proteins and Regulatable LentiviralVectors

Transduction of HeLa Cells by Inducible Lentiviral Vectors

Subconfluent HeLa cells were transduced with either HIV6X2C7SvLuc orHIV6X2C7TATALuc vector supernatant for 24 hours followed by transductionwith HIVAS521R lentiviral vector supernatant. Cells were allowed torecover from infection for 24 hours in fresh culture medium after which4-OH-tamoxifen (100 or 1000 nm) was added to the culture for anadditional 24 hours. Cells were lysed in a standard luciferase lysisbuffer, subjected to freeze thaw and analyzed for luciferase activityusing a luciferase assay kit (Promega). The results showed that cellsinfected with either HIV6X2C7SvLuc or HIV6X2C7TATALuc followed bytransduction with HIVCMVAS521R resulted in a 13.1 and 11.7 foldstimulation in luciferase activity respectively, when given4-OH-tamoxifen.

Lentiviral Transduction of Lentiviral Integrated Target VectorPopulations

HeLa cells that had been previously transduced with either HIV6X2C7SvLucor HIV6X2C7TATALuc were carried in culture for 9 passages withoutexposure to any ZFP-LBD fusion protein. On passage 10, cells weretransduced with HIVCMVAS521R for 24 hours followed by the addition of100 nm tamoxifen for an additional 24 hours. The results show that HeLacell lines containing an integrated HIV6X2C7SvLuc or HIV6X2C7TATALucvector can be induced for luciferase expression by transduction of a LVcontaining AS521R+tamoxifen 31.4- and 22.5-fold, respectively.

These data demonstrate the effectiveness of the C2H2-LBD regulator forcontrolling expression of a transgene that is stably integrated into thehost cell chromosome.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method for regulating gene expression in a cell, comprising: a)introducing into a cell a fusion protein or a nucleic acid molecule thatcomprises a sequence of nucleotides that encodes the fusion protein,wherein: a monomer of the fusion protein comprises a nucleotide bindingdomain (DBD) operatively linked to a modified ligand binding domain(LBD) from an intracellular receptor; the fusion protein is a ligandactivated transcriptional regulator; the nucleotide binding domain is apolydactyl zinc-finger domain that contains at least three modularportions thereof; each modular portion of the nucleotide binding domaininteracts with a contiguous sequence of nucleotides of at least 3nucleotides; and the ligand specificity of the LBD is modified to changeits ligand specificity compared to the ligand specificity of the ligandbinding domain of the native intracellular receptor from which the LBDwas derived, whereby ligands that activate the fusion protein are notthe ligands that activate the receptor from which the LBD was derived;and b) contacting the cell with a ligand that interacts with the bindingdomain in the fusion protein, whereby the fusion protein interacts witha target nucleic acid molecule to activate or repress transcription of agene encoded by the fusion protein.
 2. The method of claim 1, whereinthe fusion protein interacts with the target nucleic acid molecule as adimer.
 3. The method of claim 2, wherein the dimer is a heterodimer. 4.The method of claim 2, wherein the dimer is a homodimer
 5. The method ofclaim 1, wherein the fusion protein further comprises an operativelylinked transcription regulating domain.
 6. The method of claim 1,wherein the intracellular receptor is a nuclear hormone receptor.
 7. Themethod of claim 1, wherein the modified ligand-binding domain is notactivated by ligands that activate the receptor from which the LBD wasderived.
 8. The method of claim 1, wherein a module of the zinc-fingerpeptide binds to a sequence of nucleotides of the formula (GNN)n, whereG is guanidine, N is any nucleotide and n is an integer from 3 to
 6. 9.The method of claim 1, wherein the zinc-finger peptide is comprised ofmodular units from a C2H2 zinc-finger.
 10. The method of claim 1,wherein the fusion protein monomer comprises at least four zinc fingersor variants thereof.
 11. The method of claim 6, wherein the nuclearhormone receptor is selected from the group consisting of estrogenreceptors, progesterone receptors, glucocorticoid-α receptors,glucocorticoid-β receptors, mineralocorticoid receptors, androgenreceptors, thyroid hormone receptors, retinoic acid receptors, retinoidX receptors, Vitamin D receptors, COUP-TF receptors, ecdysone receptors,Nurr-1 receptors and orphan receptors.
 12. The method of claim 1,wherein the intracellular receptor is a steroid receptor.
 13. The methodof claim 6, wherein the hormone receptor is a progesterone receptorvariant or an estrogen receptor variant.
 14. The method of claim 5,wherein the transcription regulating domain comprises a transcriptionactivation domain.
 15. The method of claim 5, wherein the transcriptionregulating domain comprises a transcription activation domain selectedfrom the group consisting of VP16, VP64, TA2, STAT-6, p65 andderivatives, multimers and combinations thereof that have transcriptionactivation activity.
 16. The method of claim 14, wherein thetranscription regulating domain comprises a nuclear hormone receptortranscription activation domain or variant thereof that hastranscription activation activity.
 17. The fusion protein of claim 14,wherein the transcription regulating domain comprises a steroid hormonereceptor transcription activation domain or variant thereof.
 18. Themethod of claim 14, wherein the transcription regulating domaincomprises a viral transcription activation domain or variant thereofthat has transcription activation activity.
 19. The method of claim 18,wherein the transcription regulating domain comprises a VP16transcription activation domain or variant thereof.
 20. The method ofclaim 1, wherein the fusion protein further comprises a transcriptionregulating domain that comprises a transcription repression domain. 21.The method of claim 20, wherein the transcription repression domain isselected from the group consisting of ERD, KRAB, SID, Deacetylase, andderivatives, multimers and combinations thereof.
 22. The method of claim1, wherein the monomer of the fusion protein comprises a sequence ofamino acids encoded by the sequence of nucleotides set forth in any ofSEQ ID NOS: 1-18.
 23. The method of claim 1, wherein the target nucleicacid molecule is endogenous to the cell.
 24. The method of claim 1,wherein the target nucleic acid molecule is introduced to the cell aspart of an expression cassette.
 25. The method of claim 24, wherein theexpression cassette and fusion protein or nucleic acid encoding thefusion protein are introduced at the same time.
 26. The method of claim24, wherein the expression cassette and fusion protein or nucleic acidencoding the fusion protein are introduced sequentially.
 27. The methodof claim 1, wherein the ligand is delivered to the cell after the fusionprotein or nucleic acid molecule encoding the fusion protein isintroduced into the cell.
 28. The method of claim 1, wherein the nucleicacid molecule encoding the fusion protein comprises a vector.
 29. Themethod of claim 28, wherein the vector is a viral vector.
 30. The methodof claim 1, wherein the cell is in a mammal.
 31. The method of claim 24,wherein the expression cassette is contained in a vector.
 32. The methodof claim 31, wherein the vector is a viral vector.
 33. The method ofclaim 24, wherein the cell is in a mammal.
 34. The method of claim 1,wherein a fusion protein is introduced.
 35. The method of claim 1,wherein a nucleic acid molecule that comprises a sequence of nucleotidesencoding the fusion protein is introduced.